Primers for use in detecting beta-lactamases

ABSTRACT

Oliognucleotide primers are provided that are specific for nucleic acid characteristic of certain beta-lactamases. The primers can be employed in methods to identify nucleic acid characteristic of family-specific beta-lactamase enzymes in samples, and particularly, in clinical isolates of Gram-negative bacteria.

STATEMENT OF RELATED APPLICATIONS

This application claims the benefit of two provisional applications:Serial No. 60/102,181 filed Sep. 28, 1998, entitled “Primers for Use inDetecting Beta-Lactamases,” and Serial No. 60/121,765 filed Feb. 26,1999, also entitled “Primers for Use in Detecting Beta-Lactamases,”which are incorporated herein by reference.

BACKGROUND

A disturbing consequence of the use, and over-use, of beta-lactamantibiotics (e.g., penicillins and cephalosporins) has been thedevelopment and spread of beta-lactamases. Beta-lactamases are enzymesthat open the beta-lactam ring of penicillins, cephalosporins, andrelated compounds, to inactivate the antibiotic. The production ofbeta-lactamases is an important mechanism of resistance to beta-lactamantibiotics among Gram-negative bacteria.

Expanded-spectrum cephalosporins have been specifically designed toresist degradation by the older broad-spectrum beta-lactamases such asTEM-1, 2, and SHV-1. Microbial response to the expanded-spectrumcephalosporins has been the production of mutant forms of the olderbeta-lactamases called extended-spectrum beta-lactamases (ESBLs).Although ESBL-producing Enterobacteriaceae were first reported in Europein 1983 and 1984, ESBLs have now been found in organisms of diversegenera recovered from patients in all continents except Antarctica. Theoccurrence of ESBL-producing organisms varies widely with some typesmore prevalent in Europe (TEM-3), others more prevalent in the UnitedStates (TEM-10, TEM-12 and TEM-26), while others appear worldwide (SHV-2and SHV-5). These enzymes are capable of hydrolyzing the newercephalosporins and aztreonam. Studies with biochemical and moleculartechniques indicate that many ESBLs are derivatives of older TEM-1,TEM-2, or SHV-1 beta-lactamases, some differing from the parent enzymeby one to four amino acid substitutions.

In addition, resistance in Klebsiella pneumoniae and Escherichia coli tocephamycins and inhibitor compounds such as clavalante have also arisenvia acquisition of plasmids containing the chromosomally derived AmpCbeta-lactamase, most commonly encoded by Enterobacter cloacae,Pseudomonas aeruginosa, and Citrobacter freundii.

It is of particular concern that genes encoding the beta-lactamases areoften located on large plasmids that also contain genes for resistanceto other antibiotic classes including aminoglycosides, tetracycline,sulfonamides, trimethoprim, and chloramphenicol. Furthermore there is anincreasing tendency for pathogens to produce multiple beta-lactamases.These developments, which occur over a wide range of Gram-negativegenera, represent a recent evolutionary development in which commonGram-negative pathogens are availing themselves of increasingly complexrepertoires of antibiotic resistance mechanisms. Clinically, thisincreases the difficulty of identifying effective therapies for infectedpatients.

Thus, there is a need for techniques that can quickly and accuratelyidentify the types of beta-lactamases that may be present in a clinicalisolate or sample, for example. This could have significant implicationsin the choice of antibiotic necessary to treat a bacterial infection.

SUMMARY OF THE INVENTION

The present invention is directed to the use of oligonucleotide primersspecific to nucleic acids characteristic of (typically, genes encoding)certain beta-lactamases. More specifically, the present invention usesprimers to identify family specific beta-lactamase nucleic acids(typically, genes) in samples, particularly, in clinical isolates ofGram-negative bacteria. Specific primers of the invention include theprimer sequences set forth in SEQ ID NOs: 1-45. As used herein, anucleic acid characteristic of a beta-lactamase enzyme includes a geneor a portion thereof. A “gene” as used herein, is a segment or fragmentof nucleic acid (e.g., a DNA molecule) involved in producing a peptide(e.g., a polypeptide and/or protein). A gene can include regionspreceding (upstream) and following (downstream) a coding region (i.e.,regulatory elements) as well as intervening sequences (introns, e.g.,non-coding regions) between individual coding segments (exons). The term“coding region” is used broadly herein to mean a region capable of beingtranscribed to form an RNA, the transcribed RNA can be, but need notnecessarily be, further processed to yield an mRNA.

Additionally, a method for identifying a beta-lactamase in a clinicalsample is provided. Preferably, the clinical sample provided ischaracterized as a Gram-negative bacteria with resistance to beta-lactamantibiotics. The method includes, providing a pair of oligonucleotideprimers, wherein one primer of the pair is complementary to at least aportion of the beta-lactamase nucleic acid in the sense strand and theother primer of each pair is complementary to a different portion of thebeta-lactamase nucleic acid in the antisense strand; annealing theprimers to the beta-lactamase nucleic acid; simultaneously extending theannealed primers from a 3′ terminus of each primer to synthesize anextension product complementary to the strands annealed to each primerwherein each extension product after separation from the beta-lactamasenucleic acid serves as a template for the synthesis of an extensionproduct for the other primer of each pair; separating the amplifiedproducts; and analyzing the separated amplified products for a regioncharacteristic of the beta-lactamase.

The method, described above, can employ oligonucleotide primers that arespecific for nucleic acid of the TEM family of beta-lactamases, the K1beta-lactamases, the PSE family of beta-lactamases, and the SHV familyof beta-lactamases. Additional primers that can be used include thosethat are specific for nucleic acid of the AmpC beta-lactamases found inEnterobacter cloacae, Citrobacter freundii, Serratia marcescens,Pseudomonas aeruginosa, and E. coli.

Still other oligonucleotide primers that are suitable for use in themethod of the present invention include primers that are specific fornucleic acid of the plasmid-mediated AmpC beta-lactamases designated asFOX-1, FOX-2, or MOX-1; primers specific for nucleic acid of the OXA-9beta-lactamase; primers specific for nucleic acid of the OXA-12beta-lactamase; primers specific for the nucleic acid group of OXAbeta-lactamases representing OXA-5, 6, 7, 10, 11, 13, and 14beta-lactamases; primers specific for the OXA-1 beta-lactamases; andprimers specific for nucleic acid of the group of OXA beta-lactamasesrepresenting OXA-2, 3, and 15 beta-lactamases.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention is directed to the detection of nucleic acid thatis characteristic of (e.g., at least a segment of a gene that codes for)family-specific beta-lactamase nucleic acid in samples (e.g., clinicalisolates of Gram-negative bacteria). Specifically, the present inventionis directed to the detection of beta-lactamase nucleic acid (preferably,a gene or at least a segment of a gene) using unique primers and thepolymerase chain reaction. Using the primers and methods of the presentinvention, beta-lactamases belonging to Bush groups 1 (AmpC) and 2(TEM-1, TEM-2, SHV-1, IRTs, K1), for example, can be identified.

The primers and methods of the present invention are useful for avariety of purposes, including, for example, the identification of theprimary beta-lactamase(s) responsible for resistance to third generationcephalosporins among Gram-negative bacteria such as Escherichia coli andKlebsiella pneumoniae (Thomson et al., Antimicrob. Agents Chemother36(9):1877-1882 (1992)). Other sources of beta-lactamases include, forexample, a wide range of Enterobacteriaceae, including Enterobacterspp., Citrobacter freundii, Morganella morganii, Providencia spp., andSerratia marcescens (Jones, Diag. Microbiol. Infect. Disease31(3):461-466 (1998)). Additional beta-lactamase gene sources includePseudomonas aeruginosa (Nordmann et al., Antimicrob. Agents Chemother37(5):962-969 (1993)); Proteus mirabilis (Bret et al., Antimicrob.Agents Chemother 42(5):1110-1114 (1998)); Yersinia enterocolitica(Barnaud et al., FEMS Microbiol. Letters 148(1):15-20 (1997)); andKlebsiella oxytoca ( Marchese et al., Antimicrob. Agents Chemother42(2):464-467 (1998)).

The methods of the present invention involve the use of the polymerasechain reaction sequence amplification method (PCR) using novel primers.U.S. Pat. No. 4,683,195 (Mullis et al.) describes a process foramplifying, detecting, and/or cloning nucleic acid. Preferably, thisamplification method relates to the treatment of a sample containingnucleic acid (typically, DNA) of interest from bacteria, particularlyGram-negative bacteria, with a molar excess of an oligonucleotide primerpair, heating the sample containing the nucleic acid of interest toyield two single-stranded complementary nucleic acid strands, adding theprimer pair to the sample containing the nucleic acid strands, allowingeach primer to anneal to a particular strand under appropriatetemperature conditions that permit hybridization, providing a molarexcess of nucleotide triphosphates and polymerase to extend each primerto form a complementary extension product that can be employed inamplification of a desired nucleic acid, detecting the amplified nucleicacid, and analyzing the amplified nucleic acid for a size specificamplicon (as indicated below) characteristic of the specificbeta-lactamase of interest. This process of heating, annealing, andsynthesizing is repeated many times, and with each cycle the desirednucleic acid increases in abundance. Within in a short period of time,it is possible to obtain a specific nucleic acid, e.g., a DNA molecule,that can be readily purified and identified.

The oligonucleotide primer pair includes one primer that issubstantially complementary to at least a portion of a sense strand ofthe nucleic acid and one primer that is substantially complementary toat least a portion of an antisense strand of the nucleic acid. Theprocess of forming extension products preferably involves simultaneouslyextending the annealed primers from a 3′ terminus of each primer tosynthesize an extension product that is complementary to the nucleicacid strands annealed to each primer wherein each extension productafter separation from the beta-lactamase nucleic acid serves as atemplate for the synthesis of an extension product for the other primerof each pair. The amplified products are preferably detected by sizefractionization using gel electrophoresis. Variations of the method aredescribed in U.S. Pat. No. 4,683,194 (Saiki et al.). The polymerasechain reaction sequence amplification method is also described by Saikiet al., Science, 230, 1350-1354 (1985) and Saiki et al., Nature 324,163-166 (1986).

An “oligonucleotide,” as used herein, refers to a polymeric form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides. The term oligonucleotide refers particularly tothe primary structure, and thus includes double and single-stranded DNAmolecules and double and single-stranded RNA molecules. A “primer,” asused herein, is an oligonucleotide that is complementary to at least aportion of nucleic acid of interest and, after hybridization to thenucleic acid, may serve as a starting-point for the polymerase chainreaction. The terms “primer” or “oligonucleotide primer,” as usedherein, further refer to a primer, having a nucleotide sequence thatpossess a high degree of nucleic acid sequence similarity to at least aportion of the nucleic acid of interest. “High degree” of sequencesimilarity refers to a primer that typically has at least about 80%nucleic acid sequence similarity, and preferably about 90% nucleic acidsequence similarity. Sequence similarity may be determined, for example,using sequence techniques such as GCG FastA (Genetics Computer Group,Madison, Wisconsin), MacVector 4.5 (Kodak/IBI software package) or othersuitable sequencing programs or methods known in the art.

The terms “complement” and “complementary” as used herein, refer to anucleic acid that is capable of hybridizing to a specified nucleic acidmolecule under stringent hybridization conditions. Stringenthybridization conditions include, for example, temperatures from about50° C. to about 65° C., and magnesium chloride (MgCl₂) concentrationsfrom about 1.5 millimolar (mM) to about 2.0 mM. Thus, a specified DNAmolecule is typically “complementary” to a nucleic acid if hybridizationoccurs between the specified DNA molecule and the nucleic acid.“Complementary,” further refers to the capacity of purine and pyrimidinenucleotides to associate through hydrogen bonding in double strandednucleic acid molecules. The following base pairs are complementary:guanine and cytosine; adenine and thymine; and adenine and uracil.

As used herein, the terms “amplified molecule,” “amplified fragment,”and “amplicon” refer to a nucleic acid molecule (typically, DNA) that isa copy of at least a portion of the nucleic acid and its complementarysequence. The copies correspond in nucleotide sequence to the originalmolecule and its complementary sequence. The amplicon can be detectedand analyzed by a wide variety of methods. These include, for example,gel electrophoresis, single strand conformational polymorphism (SSCP),restriction fragment length polymorphism (RFLP), capillary zoneelectrophoresis (CZE), and the like. Preferably, the amplicon can bedetected, and hence, the type of beta-lactamase identified, using gelelectrophoresis and appropriately sized markers, according to techniquesknown to one of skill in the art.

The primers are oligonucleotides, either synthetic or naturallyoccurring, capable of acting as a point of initiating synthesis of aproduct complementary to the region of the DNA molecule containing thebeta-lactamase of interest. The primer includes nucleotides capable ofhybridizing under stringent conditions to at least a portion of at leastone strand of a nucleic acid molecule of a given beta-lactamase.Preferably, the primers of the present invention typically have at leastabout 15 nucleotides. Preferably, the primers have no more than about 35nucleotides, and more preferably, no more than about 22 nucleotides. Theprimers are chosen such that they preferably produce a primed product ofabout 200-1100 base pairs.

Optionally, a primer used in accordance with the present inventionincludes a label constituent. The label constituent can be selected fromthe group of an isotopic label, a fluorescent label, a polypeptidelabel, and a dye release compound. The label constituent is typicallyincorporated in the primer by including a nucleotide having the labelattached thereto. Isotopic labels preferably include those compoundsthat are beta, gamma, or alpha emitters, more preferably isotopic labelsare selected from the group of ³²P, 35S, and 125I. Fluorescent labelsare typically dye compounds that emit visible radiation in passing froma higher to a lower electronic state, typically in which the timeinterval between adsorption and emission of energy is relatively short,generally on the order of about 10⁻⁸ to about 10⁻³ second. Suitablefluorescent compounds that can be utilized include fluorescien andrhodamine, for example. Suitable polypeptide labels that can be utilizedin accordance with the present invention include antigens (e.g., biotin,digoxigenin, and the like) and enzymes (e.g., horse radish peroxidase).A dye release compound typically includes chemiluminescent systemsdefined as the emission of absorbed energy (typically as light) due to achemical reaction of the components of the system, includingoxyluminescence in which light is produced by chemical reactionsinvolving oxygen.

Preferred examples of these primers, that are specific for certainbeta-lactamases, are as follows, wherein “F” in the designations of theprimers refers to a 5′ upstream primer and “R” refers to a 3′ downstreamprimer. For those beta-lactamases that have more than one upstreamprimer and more than one downstream primer listed below as preferredprimers, various combinations can be used. Typically, hybridizationconditions utilizing primers of the invention include, for example, ahybridization temperature of about 50° C. to about 60° C., and a MgCl₂concentration of about 1.5 mM (millimolar) to about 2.0 mM. Althoughlower temperatures and higher concentrations of MgCl₂ can be employed,this may result in decreased primer specificity.

The following primers are specific for nucleic acid characteristic ofthe TEM family of beta-lactamase enzymes.

Primer Name: TEMprime2R

Primer Sequence: 5′-TGC TTA ATC AGT GAG GCA CC-3′ (SEQ ID NO:1)

Primer Name: TEMprime1F

Primer Sequence: 5′ -AGA TCA GTT GGG TGC ACG AG -3′ (SEQ ID NO:2)

Primer Name: TEMprimeEndR

Primer Sequence: 5′-CTT GGT CTG ACA GTT ACC-3′ (SEQ ID NO:3)

Primer Name: TEMprime2F

Primer Sequence: 5′ -TGT CGC CCT TAT TCC-3′ (SEQ ID NO:4)

Primer Name: TEMprime15F

Primer Sequence: 5′-TCG GGG AAA TGT GCG-3′ (SEQ ID NO:5)

Employing a primer pair containing the primer sequences of SEQ ID NO:1and SEQ ID NO:2 to a sample known to contain a TEM familybeta-lactamase, a size-specific amplicon of 750 base pairs willtypically be obtained. Employing a primer pair containing the primersequences of SEQ ID NO:3 and SEQ ID NO:5 to a sample known to contain aTEM family beta-lactamase, a size-specific amplicon of 992 base pairswill typically be obtained.

The following primers are specific for nucleic acid characteristic ofthe SHV family of beta-lactamase enzymes.

Primer Name: SHVprime3R

Primer Sequence: 5′ -ATC GTC CAC CAT CCA CTG CA-3′ (SEQ ID NO:6)

Primer Name: SHVprime2F

Primer Sequence: 5′-GGG AAA CGG AAC TGA ATG AG-3′ (SEQ ID NO:7)

Primer Name: SHVprime1R

Primer Sequence: 5′ -TAG TGG ATC TTT CGC TCC AG-3′ (SEQ ID NO:8)

Primer Name: SHVprime4R

Primer Sequence: 5′ -GCT CTG CTT TGT TAT TC-3′ (SEQ ID NO:9)

Primer Name: SHVprime1F

Primer Sequence: 5′ -CAC TCA AGG ATG TAT TGT G-3′ (SEQ ID NO:10)

Primer Name: SHVprimeEndR

Primer Sequence: 5′ -TTA GCG TTG CCA GTG CTC G-3′ (SEQ ID NO:11)

Employing a primer pair containing the primer sequences of SEQ ID NO:7and SEQ ID NO:11 to a sample known to contain a SHV familybeta-lactamase, a size-specific amplicon of 383 base pairs willtypically be obtained.

The following primers are specific for nucleic acid characteristic ofthe AmpC beta-lactamase enzyme (both chromosomal and plasmid-mediated)found in Enterobacter cloacae.

Primer Name: EcloC3R

Primer Sequence: 5′ -GGA ACA GAC TGG GCT TTC ATC-3′ (SEQ ID NO:12)

Primer Name: EcloC4F

Primer Sequence: 5′ -GGA CAT CCC CTT GAC-3′ (SEQ ID NO:13)

Primer Name: EcloC2R

Primer Sequence: 5′ -GTG GAT TCA CTT CTG CCA CG-3′ (SEQ ID NO:14)

Primer Name: EcloC1F

Primer Sequence: 5′ -CTT CTG GCA TGC CCT ATG AG-3′ (SEQ ID NO:15)

Primer Name: EcloCR

Primer Sequence: 5′ -CAT GAC CCA GTT CGC CAT ATC CTG-3′ (SEQ ID NO:16)

Primer Name: EcloCF

Primer Sequence: 5′-ATT CGT ATG CTG GAT CTC GCC ACC-3′ (SEQ ID NO:17)

Primer Name: EccKF

Primer Sequence: 5′-CGA ACG AAT CAT TCA GCA CCG-3′ (SEQ ID NO:44)

Primer Name: EccKR

Primer Sequence: 5′-CGG CAA TGT TTT ACT GTA GCG CC-3′ (SEQ ID NO:45)

Employing a primer pair containing the primer sequences of SEQ ID NO:14and SEQ ID NO:15 to a sample known to contain an AmpC beta-lactamasefound in Enterobacter cloacae, a size-specific amplicon of 416 basepairs will typically be obtained. Employing a primer pair containing theprimer sequences of SEQ ID NO:16 and SEQ ID NO:17 to a sample known tocontain an AmpC beta-lactamase found in Enterobacter cloacae, asize-specific amplicon of 396 base pairs will typically be obtained.Employing a primer pair containing the primer sequences of SEQ ID NO:14and SEQ ID NO:17 to a sample known to contain an AmpC beta-lactamasefound in Enterobacter cloacae, a size-specific amplicon of 601 basepairs will typically be obtained. Employing a primer pair containing theprimer sequences of SEQ ID NO:17 and SEQ ID NO:45 to a sample known tocontain an AmpC beta-lactamase found in Enterobacter cloacae, asize-specific amplicon of 688 base pairs will typically be obtained.Employing a primer pair containing the primer sequences of SEQ ID NO:44and SEQ ID NO:45 to a sample known to contain an AmpC beta-lactamasefound in Enterobacter cloacae, a size-specific amplicon of 1529 basepairs will typically be obtained.

The following primers are specific for nucleic acid characteristic ofthe AmpC beta-lactamase enzyme (both chromosomal and plasmid-mediated)found in Citrobacter freundii. Although these primers cross-react withthe chromosomal AmpC from E. coli, the band produced from the E. coliAmpC is much larger. Thus, the primers can be used to differentiallyidentify Citrobacter from E. coli.

Primer Name: CFC1F

Primer Sequence: 5′ -CTG GCA ACC ACA ATG GAC TCC G-3′ (SEQ ID NO:18)

Primer Name: CFC1R

Primer Sequence: 5′ -GCC AGT TCA GCA TCT CCC AGC C-3′ (SEQ ID NO:19)

Employing a primer pair containing the primer sequences of SEQ ID NO:18and SEQ ID NO:19 to a sample known to contain an AmpC beta-lactamasefound in Citrobacter freundii, a size-specific amplicon of 419 basepairs will typically be obtained.

The following primers are specific for nucleic acid characteristic ofthe AmpC beta-lactamase enzyme (both chromosomal and plasmid-mediated)found in Serratia marcescens.

Primer Name: SMC1F

Primer Sequence: 5′ -CGT GAC CAA CAA CGC CCA GC-3′ (SEQ ID NO:20)

Primer Name: SMC1R

Primer Sequence: 5′-CCA GAT AGC GAA TCA GAT CGC-3′ (SEQ ID NO:21)

Employing a primer pair containing the primer sequences of SEQ ID NO:20and SEQ ID NO:21 to a sample known to contain an AmpC beta-lactamasefound in Serratia marcescens, a size-specific amplicon of 335 base pairswill typically be obtained.

The following primers are specific for nucleic acid characteristic ofthe plasmid-mediated AmpC beta-lactamase enzyme designated as FOX-1,FOX-2, MOX-1, and others in this family.

PrimerName: FOX1F

Primer Sequence: 5′ -CCA GCC GAT GCT CAA GGA G-3′ (SEQ ID NO:22)

Primer Name: FOX1R

Primer Sequence: 5′ -CAC GAA CGC CAC ATA GGC G-3′ (SEQ ID NO:23)

Employing a primer pair containing the primer sequences of SEQ ID NO:22and SEQ ID NO:23 to a sample known to contain a plasmid-mediated AmpCbeta-lactamase, such as FOX-1, FOX-2, and MOX-1, a size-specificamplicon of 937 base pairs will typically be obtained.

The following primers are specific for nucleic acid characteristic ofthe AmpC beta-lactamase enzyme (chromosomal) found in Pseudomonasaeruginosa.

Primer Name: PaerugR

Primer Sequence: 5′-GGC ATT GGG ATA GTT GCG GTT G-3′ (SEQ ID NO:24)

Primer Name: PaerugF

Primer Sequence: 5′-TTA CTA CAA GGT CGG CGA CAT GAC C-3′ (SEQ ID NO:25)

Employing a primer pair containing the primer sequences of SEQ ID NO:24and SEQ ID NO:25 to a sample known to contain an AmpC beta-lactamasefound in Pseudomonas aeruginosa, a size-specific amplicon of 268 basepairs will typically be obtained.

The following primers are specific for nucleic acid characteristic ofthe AmpC beta-lactamase enzyme (both chromosomal and plasmid-mediated)found in E. coli.

Primer Name: ECOLI C1F

Primer Sequence: 5′-GGA TCA CAC TAT TAC ATC TCG C-3′ (SEQ ID NO:26)

Primer Name: ECOLI C1R

Primer Sequence: 5′-CGT ATG GTT GAG TTT GAG TGG C-3′ (SEQ ID NO:27)

Employing a primer pair containing the primer sequences of SEQ ID NO:26and SEQ ID NO:27 to a sample known to contain an AmpC beta-lactamasefound in E. coli., a size-specific amplicon of 254 base pairs willtypically be obtained.

The following primers are specific for nucleic acid characteristic ofthe K1 beta-lactamase enzyme.

Primer Name: TOHO-1F

Primer Sequence: 5′-GCG ACC TGG TTA ACT ACA ATC CC-3′ (SEQ ID NO:28)

Primer Name: TOHO-1R

Primer Sequence: 5′-CGG TAG TAT TGC CC TTA AGC C-3′ (SEQ ID NO:29)

Primer Name: MEN-1F

Primer Sequence: 5′-CGG AAA AGC ACG TCG ATG GG-3′ (SEQ ID NO:30)

Primer Name: MEN-1R

Primer Sequence: 5′-GCG ATA TCG TTG GTG GTG CC-3′ (SEQ ID NO:31)

Employing a primer pair containing the primer sequences of SEQ ID NO:28and SEQ ID NO:29 to a sample known to contain a K1 beta-lactamase, asize-specific amplicon of 351 base pairs will typically be obtained.Employing a primer pair containing the primer sequences of SEQ ID NO:30and SEQ ID NO:31 to a sample known to contain a K1 beta-lactamase, asize-specific amplicon of 415 base pairs will typically be obtained.

The following primers are specific for nucleic acid characteristic ofthe PSE family of beta-lactamase enzymes.

PrimerName: PSE 1F

Primer Sequence: 5′-CTC GAT GAT GCG TGC TTC GC-3′ (SEQ ID NO:32)

Primer Name: PSE 1R

Primer Sequence: 5′-GCG ACT GTG ATG TAT AAA CG-3′ (SEQ ID NO:33)

Employing a primer pair containing the primer sequences of SEQ ID NO:32and SEQ ID NO:33 to a sample known to contain a PSE1, PSE4, and/or CARB3beta-lactamase, a size-specific amplicon of 523 base pairs willtypically be obtained. If a PSE2 (OXA10) beta-lactamase is present inthe sample, it is possible that some cross-reactivity with the primerpair may occur.

The following primers are specific for nucleic acid characteristic ofthe OXA-9 beta-lactamase enzyme.

Primer Name: OXA 91F

Primer Sequence: 5′-CGT CGC TCA CCA TAT CTC CC-3′ (SEQ ID NO:34)

Primer Name: OXA 91R

Primer Sequence: 5′-CCT CTC GTG CTT TAG ACC CG-3′ (SEQ ID NO:35)

Employing a primer pair containing the primer sequences of SEQ ID NO:34and SEQ ID NO:35 to a sample known to contain a OXA-9 beta-lactamase, asize-specific amplicon of 315 base pairs will typically be obtained.

The following primers are specific for nucleic acid characteristic ofthe OXA-12 beta-lactamase enzyme.

Primer Name: OXA121F

Primer Sequence: 5′-CGC TGG GAA ACC TAT TCGG-3′ (SEQ ID NO:36)

Primer Name: OXA121R

Primer Sequence: 5′-CTG CCA TCC AGT TTC TTC GGG-3′ (SEQ ID NO:37)

Employing a primer pair containing the primer sequences of SEQ ID NO:36and SEQ ID NO:37 to a sample known to contain a OXA-12 beta-lactamase, asize-specific amplicon of 341 base pairs will typically be obtained.

The following primers are specific for nucleic acid characteristic ofthe OXA-5, 6, 7, 10, 11, 13, and 14 beta-lactamase enzymes.

PrimerName: OXA711F1

Primer Sequence: 5′-GGT GGC ATT GAC AAA TTC TGG-3′ (SEQ ID NO:38)

Primer Name: OXA711B2

Primer Sequence: 5′-CCC ACC ATG CGA CAC CAG-3′ (SEQ ID NO:39)

Employing a primer pair containing the primer sequences of SEQ ID NO:38and SEQ ID NO:39 to a sample known to contain an OXA-5, 6, 7, 10, 11, 13or 14 beta-lactamase, a size-specific amplicon of 226 base pairs willtypically be obtained.

The following primers are specific for nucleic acid characteristic ofthe OXA-1 beta-lactamase enzyme.

Primer Name: OXA 1 F2

Primer Sequence: 5′-TGT GCA ACG CAA ATG GCA C-3′ (SEQ ID NO:40)

Primer Name: OXA1B14

Primer Sequence: 5′-CGA CCC CAA GTT TCC TGT AAG TG-3′ (SEQ ID NO:41)

Employing a primer pair containing the primer sequences of SEQ ID NO:40and SEQ ID NO:41 to a sample known to contain a OXA-1 beta-lactamase, asize-specific amplicon of 579 base pairs will typically be obtained.

The following primers are specific for nucleic acid characteristic ofthe OXA-2, 3, and 15 beta-lactamase enzymes.

Primer Name: OXA 23 F1

Primer Sequence: 5′-AGG CAC GAT AGT TGT GGC AGA C-3′ (SEQ ID NO:42)

Primer Name: OXA23B3

Primer Sequence: 5′-CAC TCA ACC CAT CCT ACC CAC C-3′ (SEQ ID NO:43)

Employing a primer pair containing the primer sequences of SEQ ID NO:42and SEQ ID NO:43 to a sample known to contain a OXA-2, 3 or 15beta-lactamase, a size-specific amplicon of 555 base pairs willtypically be obtained.

Various other primers, or variations of the primers described above, canalso be prepared and used according to methods of the present invention.For example, alternative primers can be designed based on targetedbeta-lactamases known or suspected to contain regions possessing highG/C content (i.e., the percentage of guanine and cytosine residues). Asused herein, a “high G/C content” in a target nucleic acid, typicallyincludes regions having a percentage of guanine and cytosine residues ofabout 60% to about 90%. Thus, changes in a prepared primer will alter,for example, the hybridization or annealing temperatures of the primer,the size of the primer employed, and the sequence of the specificresistance gene or nucleic acid to be identified. Therefore,manipulation of the G/C content, e.g., increasing or decreasing, of aprimer or primer pair may be beneficial in increasing detectionsensitivity in the method.

Additionally, depending on the suspected nucleic acid in the sample, aprimer of the invention can be prepared that varies in size. Typically,primers of the invention are about 12 nucleotides to about 50nucleotides in length, preferably the primers are about 15 nucleotidesto about 25 nucleotides in length. Oligonucleotides of the invention canreadily be synthesized by techniques known in the art (see, for example,Crea et al., Proc. Natl. Acad. Sci. (U.S.A.) 75:5765 (1978)).

Once the primers are designed, their specificity can be tested using thefollowing method. Depending on the target nucleic acid of clinicalinterest, a nucleic acid is isolated from a bacterial control strainknown to express or contain the resistance gene. This control strain, asused herein, refers to a “positive control” nucleic acid (typically,DNA). Additionally, a “negative control” nucleic acid (typically, DNA)can be isolated from one or more bacterial strains known to express aresistance gene other than the target gene of interest. Using thepolymerase chain reaction, the designed primers are employed in adetection method, as described above, and used in the positive andnegative control samples and in at least one test sample suspected ofcontaining the resistance gene of interest. The positive and negativecontrols provide an effective and qualitative (or grossly quantitative)means by which to establish the presence or the absence of the gene ofinterest of test clinical samples. It should be recognized that with asmall percentage of primer pairs, possible cross-reactivity with otherBeta-lactamase genes might be observed. However, the size and/orintensity of any cross-reactive amplified product will be considerablydifferent and can therefore be readily evaluated and dismissed as anegative result.

The invention also relates to kits for identifying a family specificbeta-lactamase enzymes by PCR analysis. Kits of the invention typicallyinclude one or more primer pairs specific for a beta-lactamase ofinterest, one or more positive controls, one or more negative controls,and protocol for identification of the beta-lactamase of interest usingpolymerase chain reaction. A negative control includes a nucleic acid(typically, DNA) molecule encoding a resistant beta-lactamase other thatthe beta-lactamase of interest. The negative control nucleic acid may bea naked nucleic acid (typically, DNA) molecule or inserted into abacterial cell. Preferably, the negative control nucleic acid is doublestranded, however, a single stranded nucleic acid may be employed. Apositive control includes a nucleic acid (typically, DNA) that encodes abeta-lactamase from the family of beta-lactamases of interest. Thepositive control nucleic acid may be a naked nucleic acid molecule orinserted into a bacterial cell, for example. Preferably, the positivecontrol nucleic acid is double stranded, however, a single strandednucleic acid may be employed. Typically, the nucleic acid is obtainedfrom a bacterial lysate.

Accordingly, the present invention provides a kit for characterizing andidentifying a family specific beta-lactamase that would have generalapplicability. Preferably, the kit includes a polymerase (typically, DNApolymerase) enzyme, such as Taq polymerase, and the like. A kit of theinvention also preferably includes at least one primer pair that isspecific for a beta-lactamase. A buffer system compatible with thepolymerase enzyme is also included and are well known in the art.Optionally, the at least one primer pair may contain a labelconstituent, a fluorescent label, a polypeptide label, and a dye releasecompound. The kit may further contain at least one internal samplecontrol, in addition to one or more further means required for PCRanalysis, such as a reaction vessel. If required, a nucleic acid fromthe bacterial sample can be isolated and then subjected to PCR analysisusing the provided primer set of the invention.

In another embodiment, family specific beta-lactamase enzymes inclinical samples, particularly clinical samples containing Gram-negativebacteria, can be detected by the primers described herein in a“microchip” detection method. In a microchip detection method, nucleicacid, e.g., genes, of multiple beta-lactamases in clinical samples canbe detected with a minimal requirement for human intervention.Techniques borrowed from the microelectronics industry are particularlysuitable to these ends. For example, micromachining andphotolithographic procedures are capable of producing multiple parallelmicroscopic scale components on a single chip substrate. Materials canbe mass produced and reproducibility is exceptional. The microscopicsizes minimize material requirements. Thus, human manipulations can beminimized by designing a microchip type surface capable of immobilizinga plurality of primers of the invention on the microchip surface.

Thus, an object of the present invention is to provide a parallelscreening method wherein multiple serial reactions are automaticallyperformed individually within one reaction well for each of theplurality of nucleic acid strands to be detected in the plural parallelsample wells. These serial reactions are performed in a simultaneous runwithin each of the multiple parallel lanes of the device. “Parallel” asused herein means wells identical in function. “Simultaneous” meanswithin one preprogrammed run. The multiple reactions automaticallyperformed within the same apparatus minimize sample manipulation andlabor.

Thus, the present invention provides multiple reaction wells, thereaction wells being reaction chambers, on a microchip, each reactionwell containing an individualized array to be used for detecting abeta-lactamase gene uniquely specified by the substrates provided, thereaction conditions and the sequence of reactions in that well. The chipcan thus be used as a method for identifying beta-lactamase genes inclinical samples.

Objects and advantages of this invention are further illustrated by thefollowing examples, but the particular materials and amounts thereofrecited in these examples, as well as other conditions and details,should not be construed to unduly limit this invention.

EXAMPLE 1 Klebsiella pneumoniae with ESBLs and a Plasmid-mediated AmpCBeta-lactamase

Materials and Methods

Klebsiella pneumoniae 225

Klebsiella pneumoniae 225 was isolated from a 43-year-old white malepatient who was working in a New York City sewage canal on Apr. 26,1996. While in a pit containing a layer of sewage, a screen fell,knocking the patient down in the sewage. The patient struck his leftforehead, sustaining a severe laceration approximately 20 centimeters(cm) long and down to the skull and was unconscious for approximatelyfive minutes. The patient was taken to a local hospital where the woundwas surgically debrided, irrigated, and closed. Intravenous cefazolinand gentamicin were administered pre-operatively, and cefazolin wascontinued 24 hours post-operatively. The patient was discharged andreturned to Omaha, Nebr., four days later.

On May 2, 1996, the patient was seen by an Omaha surgeon who diagnosedwound infection, ordered culture of the wound drainage, and initiatedtherapy with cephalexin and penicillin. The culture yielded growth of K.pneumoniae, Aeromonas hydrophila and Proteus penneri. By May 24, 1996,the patient was experiencing significant swelling and pain in the leftscalp area, significant weakness and dizziness, and a low grade fever.An infectious disease consult was ordered, and the patient washospitalized for further evaluation and management.

Laboratory findings included a white blood cell count of 21,000 percubic millimeter (cmm). Aspiration of a bulging left temporal mass fromthe patient yielded 7 milliliter (ml) of purulent fluid from which K.pneumoniae and A. hydrophila were cultured.

The patient was empirically treated with piperacillin/tazobactam andciprofloxacin. On May 25, 1996, the patient had incision drainage anddebridement of the wound. Operative findings as well as preoperative CTscan of the patient's head did not reveal osteomyelitis. There was anabscess commencing in the region of the left zygoma and extendingsuperior to the parietal region, with two small opaque foreign bodies inthe caudal aspect of the collection. On May 27, 1996, followingantibiotic susceptibility results, therapy was changed toimipenem/cilastatin. The drain was removed and the patient wasdischarged on May 29, 1996, and treated at home with intravenousimipenem/cilastatin via a peripheral inserted central catheter. Afterfour weeks of therapy all signs of inflammation resolved. The patientremained free from infection at follow-up on Dec. 10, 1996.

Susceptibility Tests

Susceptibility tests were performed by microdilution methodology usingthe MicroScan Walkaway system (Dade MicroScan Inc., Sacramento, Calif.)and by NCCLS microdilution methodology in Mueller-Hinton broth (CM 405,Oxoid, Basingstoke, England) using an inoculum of approximately 5×10⁵CFU/ml (National Committee for Clinical Laboratory Standards, 1997,Approved Standard M7-A4) and also by NCCLS disk diffusion methodology(National Committee for Clinical Laboratory Standards, 1997, ApprovedStandard M2-A6).

Clavulanate Double-Disk Potentiation Test

Using the procedure of Brun-Buisson et al., Lancet., ii 302-306 (1987),a Mueller-Hinton agar plate (CM 337, Oxoid, Basingstoke, England) wasinoculated with K. pneumoniae 255 as for a standard disk diffusion test.Disks (BBL, Cockeysville, Md.) containing aztreonam, cefotaxime,ceftriaxone, and ceftazidime were strategically placed around anamoxicillin-clavulanate disk prior to incubation at 35° C. ESBLproduction was inferred by the presence of characteristic distortions ofthe inhibition zone indicative of clavulanate potentiation of the testdrug.

Three-Dimensional Test

Using a modification of the procedure of Thomson and Sanders,

Antimicrob. Agents Chemother., 36:1877-1882 (1992), the surface of aMueller-Hinton agar plate was inoculated with E. coli ATCC 25922 as fora standard disk diffusion test. A slit made in the agar with a sterileno. 11 scalpel blade was then inoculated with a heavy suspension ofcells of K. pneumoniae 225 that had been grown to logarithmic phase in10 ml tryptone soy broth (CM 129, Oxoid), centrifuged, and resuspendedin 100 microliter (μl) TRIS EDTA buffer (T-9285 Sigma Chemical Co., St.Louis, Mo.) for 40 minutes. Disks containing aztreonam, cefotaxime,ceftriaxone, ceftazidime and cefoxitin were placed on the agar 3millimeters (mm) away from the inoculated slit, and the plate wasincubated in the usual manner.

Enzymatic inactivation of the antibiotics was inferred if the margin ofthe inhibition zone was distorted in the vicinity of the slit in amanner that indicated loss of drug activity (hydrolysis) as the drugdiffused through the inoculated slit.

Isoelectric Focusing, Cefotaxime Hydrolysis, and InhibitorDeterminations

Using a modification of the methods of Sanders et al., Antimicrob.Agents Chemother., 30:951-952 (1986), Bauernfeind et al., Infection, 18;294-298 (1990), and Thomson et al., Antimicrob. Agents Chemother.,35;1001-1003 (1991), sonic extracts of K. pneumoniae 225 and strains ofE. coli that produced reference beta-lactamases, were characterized bydetermining the isoelectric focusing point (pI) of each beta-lactamase,inhibitor profile in the presence and absence of 1,000 micromolar (μM)clavulanate and 1,000 μM cloxacillin, and ability to hydrolyze 0.75μg/ml cefotaxime solution.

Plasmid Isolations

Plasmid DNA isolated using alkaline lysis (Manniatis et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1982) wasperformed with the following modifications. Cell pellets were washedtwice with 3% TRITON-X 100 dissolved in Tris-ethylenediaminetetraacetate (Tris-EDTA) (pH 8). After neutralization,supernatant was extracted with phenol plus 1/10 volume 10% sodiumdodecyl sulfate (SDS) followed by one extraction usingphenol:chloroform:isoamyl alcohol (25:24:1) followed by one or morechloroform:isoamyl (24:1) extractions until supernatant was clear. Whendesignated, some samples were treated with plasmid-safe DNase (EpicentreTechnologies, Madison, Wis.) as described by the manufacturer. PlasmidDNA was electrophoresed on the day of preparation to decrease thepossibility of DNA damage (nicking) during storage. Plasmids wereseparated by agarose (0.8 %) gel electrophoresis using 1×Tris-acetate-EDTA (TAE) as the buffer system.

In some cases, plasmids were visualized without previous isolation bylysing the bacterial cells within the well of the agarose gel. Onecolony of Klebsiella pneumoniae 225 was suspended into 5 μl ofprotoplasting buffer (30 mM Tris-HCl (pH 8), 5 mM EDTA, 50 mM NaCl, 20%weight by volume (w/v) sucrose, 50 μg/ml RNase A and 50 μg/ml lysozyme;the RNase A and lysozyme added just prior to use) and incubated 30minutes at 37° C. Into each well of the agarose gel, 2 μl of roomtemperature lysis buffer (89 mM Tris (pH 8.3), 89 mM boric acid, 25 mMEDTA, 2% w/v SDS, 5% w/v sucrose and 0.04% Bramaphenol blue) was loadedjust prior to the addition of protoplast suspension. The protoplastsuspension was loaded and the gel was run for 15 minutes at 30 volts tolyse the protoplasts. After 15 minutes the voltage was increased to 120volts and the gel was run for 1-1.5 hours. Before staining in ethidiumbromide (0.5 μg/ml), the gel was washed in large volumes of water withat least two changes to remove the SDS. The plasmid bands werevisualized with a UV transilluminator. The gel consisted of 4.8%agarose, 1× Tris-borate-EDTA (TBE) (89 mM Tris, 89 mM boric acid, and2.5 mM EDTA) (pH 8.3) and 10% SDS. The running buffer was 1× TBE plus10% SDS.

Southern Analysis

Plasmid DNA was prepared by alkaline lysis separated as described above.To achieve high resolution separation, gels were electrophoresed for17-18 hours at 35 volts. DNA was transferred using 0.4 M NaOH toZeta-Probe blotting membranes using a vacuum blotter (Bio-RAD) asdescribed by the manufacturer. TEM specific probes(5′-TGCTTAATCAGTGAGGCACC-3′ (SEQ ID NO:1) nucleotides 1062-1042;numbering of Sutcliff, Proc. Nat. Aca. Sci. USA, 75:3737-3741 (1978))and SHV (5′-TTAGCGTTGCCAGTGCTCG-3′ (SEQ ID NO:11 nucleotides 988-970;numbering of Mercier et al., Antimicrob. Agents Chemother., 34:1577-1583(1990)) were labeled using the Genius System Oligonucleotide 3′-Endlabeling kit (Boehringer Mannheim, Indianapolis, Ind.). Prehybridizationand hybridization followed the recommendation of the manufacturer using1% SDS at 37° C. Initially, blots were washed with 5× SSC (twice at roomtemperature for 5 minutes and twice at room temperature for 30 minutesfollowed by washings using tetramethylammonium chloride (TMAC); once at37° C. for 15 minutes and twice at 48° C. for 20 minutes. Labeled probehybridized to plasmid DNA was detected using the Genius Luminescentdetection kit (Boehringer Mannheim) as described by manufacturer.

Polvmerase Chain Reaction (PCR) Template was prepared as below inExample 3. Primers used for amplification are listed in Table 1.

TABLE 1 PCR Primers Beta-lactamase Gene Sequence (nucleotide, nt) TEM¹(Forward) 5′-AGATCAGTTGGGTGCACGAG-3′ (nt 313-332) (SEQ ID NO:2)(Reverse) 5′-TGCTTAATCAGTGAGGCACC-3′ (nt 1061-1042) (SEQ ID NO:1) SHV²(Forward) 5′-GGGAAACGGAACTGAATGAG-3′ (nt 606-625) (SEQ ID NO:7)(Reverse) 5′-ATCGTCCACCATCCACTGCA-3′ (nt 757-738) (SEQ ID NO:6) OXA-9³(Forward) 5′-CGTCGCTCACCATATCTCCC-3′ (nt 2783-2802) (SEQ ID NO:34)(Reverse) 5′-CCTCTCGTGCTTTAGACCCG-3′ (nt 3097-3078) (SEQ ID NO:35)Enterobacter (Forward) 5′-ATTCGTATGCTGGATCTCGCCACC-3′ AmpC⁴ (nt 413-436)(SEQ ID NO:17) (Reverse) 5′-CATGACCCAGTTCGCCATATCCTG-3′ (nt 808-785)(SEQ ID NO: 16) ¹Sequence Reference = Sutcliffe et al., Proc. Nat. Aca.Sci. USA, 75, 3737-3741 (1978). ²Sequence Reference = Mercier et al.,Antimicrob. Agents Chemother., 34, 1577-1583 (1990). ³Sequence Reference= Tolmasky et al., Plasmid, 24, 218-226 (1990). ✓⁴Sequence Reference =Galleni et al., Biochem. J., 250, 753-760 (1988).

PCR amplifications were carried out as described below in Example 3 withthe following modifications. The composition of the reaction mixture was10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 0.2 mM (each) of thefour deoxynucleoside triphosphates, and 1.2 U of Taq polymerase (GIBCO,Gaithersburg, Md.) in a total volume of 48 μl. A total of 2 μl of samplelysate containing the DNA template was added to the reaction mixture.The PCR parameters consisted of initial denaturation step at 95° C. for5 minutes followed by 24 amplification cycles consisting of adenaturation step of 96° C. for 15 seconds; primer annealing at 55° C.for 15 seconds and extension at 72° C. for 2 minutes. Amplified productwas detected by agarose (2%) gel electrophoresis using a 1× TAEbuffering system. Some of the PCR products were sequenced by automatedPCR cycle-sequencing with dye-terminator chemistry using a DNA stretchsequencer from Applied Biosystems (Foster City, Calif.).

Restriction Fragment Length Polymorphism (RFLP)

SHV-specific PCR products (1/5 volume) were used directly in arestriction endonuclease assay (Niesch-Inderbinen et al., Eur. J. Clin.Microbiol. Infect. Dis., 15:398-402 (1996)) using the restrictionendonuclease, NheI (New England Biolabs, Beverly, Mass.). Enzymereactions were carried out as directed by the manufacturer. To ensurethat each sample received the same amount of enzyme, an enzyme mixcontaining the buffer system and enzyme was aliquoted to each sample.Samples were resolved using 2% agarose and a 1× TAE buffer system.

Transformation and Conjugation

Transformations were done using a modified Hanahan method (TSS)described by CLONTECH (CLONTECH Laboratories, Inc., Palo Alto, Calif.,Transformer site. Directed Mutagenesis Kit—2nd version). Plasmids wereseparated as described above, excised from the gel, and electroelutedfrom the gel slice. The DNA was transformed into E. coli HB101.

Conjugation experiments were carried out by filter mating using E. coli,strain C600, as the recipient. Transconjugants were selected onLuria-Bertani agar plates containing 30 μg/ml of naladixic acid. Anendol test was performed on the transconjugant (E. coli C600) to furtherdifferentiate it from the donor (K. pneumoniae 225).

Results

Susceptibility Tests

The results of the microdilution tests performed with K. pneumoniae 225using the NCCLS microdilution methodology were as follows:

MIC>64 μg/ml: ticarcillin, ticarcillin/clavulanate, piperacillin,piperacillin/tazobactam, ceftazidime, cefixime, loracarbef, cephalothin,cefazolin, cefoxitin, aztreonam, ampicillin/sulbactam

MIC 64 μg/ml: amoxicillin/clavulanate, cefpodoxime

MIC 16 μg/ml: ceftriaxone, cefotaxime

MIC 1 μg/ml: imipenem, cefepime

MIC 0.5 μg/ml: ciprofloxacin

MIC 0.06 μg/ml: meropenem

Other susceptibility results obtained in MicroScan tests were (MicroScanMICs shown in parentheses):

Resistant: cefuroxime (>16 μg/ml), gentamicin (>8 μg/ml), tobramycin (>8μg/ml), amikacin (>32 μg/ml), trimethoprim/sulfamethoxazole (>2/38),tetracycline (>8 μg/ml), nitrofurantoin (>64 μg/ml), chloramphenicol(>16 μg/ml)

Susceptible: ofloxacin and levofloxacin (both 2 μg/ml), cefotetan (16μg/ml)

Discrepancies between MicroScan and conventional NCCLS results wereobtained with ciprofloxacin (0.5 μg/ml in conventional microdilutiontest, susceptible by disk test, 2 μg/ml in MicroScan test) and cefotetan(resistant in disk test with 12 mm zone diameter, susceptible byMicroScan, 16 μg/ml).

The susceptibility results for the Aeromonas hydrophila isolate were notconsidered unusual and are not reported.

Double Disk and Three Dimensional Tests

The clavulanate double-disk potentiation test was positive with each ofthe antibiotics tested, indicating that K. pneumoniae possessed one ormore clavulanate-sensitive beta-lactamases capable of hydrolyzingaztreonam, cefotaxime, ceftriaxone, and ceftazidime. This result wasconsistent with beta-lactamase activity of Bush group 2be or, possibly,high level activity of Bush group 2b.

The three dimensional test was positive for each of the antibioticstested, aztreonam, cefotaxime, ceftriaxone, ceftazidime, and cefoxitin,indicating β-lactamase-mediated hydrolysis of each drug. The positiveresult with cefoxitin was notable, being consistent with production of aBush group 1 beta-lactamase.

Isoelectric focusing-Based Tests

Isoelectric focusing yielded five beta-lactamase bands with pI values of5.4, 6.8, 7.6, 8.2, and ³9.0, values consistent with TEM-1 (pI 5.4),PSE-3, OXA-9 or unknown enzyme (pI 6.8), SHV-1, SHV-2, or SHV-8 (pI7.6), SHV-5 (pI 8.2) and AmpC (pI ³9.0) (Table 2, below). Only the pI³9.0 enzyme was resistant to clavulanate, confirming that this was aBush group 1 (AmpC) beta-lactamase. The beta-lactamase bands whichhydrolyzed cefotaxime, as detected by microbiological assay, were pI7.6, pI 8.2, and pI ³9.0. These results suggested the presence ofclavulanate-sensitive ESBLs of pI values 7.6 and 8.2, and added supportto the identification of an AmpC enzyme with a pI value ³9.0. Theseresults are supported and/or confirmed by PCR (see below).

TABLE 2 Isoelectric Focusing pI Ca Sensitive Ctx Hydrolyzed PossibleEnzyme 5.4 S − TEM-1 6.8 S − OXA-9 7.6 S + SHV-1, SHV-2 8.2 S +/− SHV-5˜9.3 R + AmpC Ca = clavulanate (1000 μm) Ctx = cefotasime (0.75 μg/ml)

Polymerase Chain Reaction (PCR)

PCR analysis was used initially to confirm and/or identify thebeta-lactamases observed during isoelectric focusing (Table 2, above).Primer sets specific for the TEM or SHV gene families, EnterobacterAmpC, OXA-9 and integron sequences were used in a PCR (Table 1, above).PCR identified the presence of TEM and SHV-like genes, an EnterobacterAmpC-like gene, the OXA-9 gene and integron sequences (data not shown).

Plasmids

Multiple plasmid isolations from K. pneumoniae 225 revealed the organismcarried only two plasmids. Using a supercoiled DNA ladder, the estimatedsizes of these plasmids were approximately 17 kb and approximately 90kb. Three different isolation procedures were used to extract plasmidDNA; alkaline lysis (Manniatis et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor, N.Y., 1982), lysozymes/SDS (Crosa et al.,“Plasmids” In Gerhardt et al., Manual of Methods for GeneralBacteriology, American Society for Microbiology, Washington, DC, pages266-282, 1981), and cell lysis and extraction within the well of thegel. All procedures yielded the same two plasmids indicating thatplasmids were not being lost during any one type of isolation procedure.Alkaline lysis yielded the purest preparation of plasmid DNA and wastherefore used for southern blot analysis. It was possible that residualchromosomal DNA comigrated and therefore masked a possible plasmid. Toaddress this, an enzyme, plasmid-safe DNase (Epicentre Technologies,Madison, Wis.), which does not cleave supercoiled DNA, was used to treatthe DNA plasmid preparations before electrophoresis. Treatment withplasmid-safe DNase degraded the chromosomal DNA band while having noeffect on the 17 kb and 90 kb plasmids, indicating no other plasmidswere present in K. pneumoniae 225 (data not shown).

Southern Analysis

It was surprising that an organism expressing 5 and possibly 6beta-lactamases would have only two extrachromosomal pieces of DNA.Therefore, whether beta-lactamase genes were encoded on one or bothplasmids was evaluated. Southern analysis revealed that both the 90 kband 17 kb plasmid encoded TEM-like genes, however, only the 90 kbplasmid encoded the SHV-like genes (data not shown).

Transformation and Conjugation

It was possible that the plasmid-mediated AmpC gene encoded by K.pneumoniae 225 cross-hybridized with the TEM-specific probe and that oneor both plasmids encoded the AmpC enzyme observed during isoelectricfocusing. In an attempt to isolate each plasmid from the other,transformation experiments were carried out. Each plasmid was extractedand gel purified. The approximately 17 kb plasmid was transformed intoE. coli HB101, and selected using ampicillin. After confirming that onlya 17 kb plasmid was present in the HB 101 transformants, a diskdiffusion assay was performed (Table 3).

TABLE 3 Disk Diffusion Assay Zone Size (mm) Drug Kleb 225 Tr (17 kb) TcChloramphenical 8 (R) 22 (S) 12 (R) Gentomicin 8 (R) 25 (S) 8 (R)Cefotetan 12 (R) 30 (S) 11 (R) Ceftriaxone 13 (R) 32 (S) 12 (R)Cefotaxime 15 (I) 34 (S) 12 (R) Ceftazidime 8 (R) 33 (S) 7 (R)Pippercillin/Tazpbactam 15 (R) 30 (S) 14 (R) Trimethoprim/ 6 (R) 31 (S)8 (R) Sulfamethoxazole Cefoxitin 6 (R) 25 (S) 7 (R) Aztreonam 8 (R) 33(S) 7 (R) Ciprofloxacin 22 (S) 31 (S) 31 (S) Imipenem 22 (S) 33 (S) 23(S) Amikacin — 12 (R) — Ampicillin — 7 (R) — Tr = transformant Tc =transconjugate

The transformant did not exhibit diminished susceptibility to any of thedrugs in Table 3 except ampicillin and amikacin, indicating that the 17kb plasmid did not encode AmpC or extended spectrum beta-lactamasegenes. Several attempts to transform the large plasmid into E. coli,(strains HB101 and MV1190) failed. Transformation using the 90 kbplasmid produced transformants that were resistant only to ampicillin.When plasmid DNA was isolated from these transformants many sizedplasmids, all less than 90 kb, were present (data not shown).

The data obtained from the transformation of the 17 kb plasmid suggestedthat the plasmid-mediated AmpC gene was encoded on the 90 kb plasmid.Therefore, conjugation experiments were performed. Conjugation betweenK. pneumoniae 225 and E. coli C600 resulted in the transfer of both the90 kb and 17 kb plasmids. Cefoxitin resistance of the transconjugantindicated transfer of the AmpC gene (Table 3). Taken together, thesedata strongly suggest that the AmpC gene is located on the 90 kbplasmid.

Restriction Fragment Length Polymorphism (RFLP)

Some ESBL-SHV enzymes with a pI of 7.6 (SHV-2, SHV-7) contain a glycineto serine amino acid substitution at position 238. In the structuralSHV-gene the nucleotide mutation resulting in the amino acidsubstitution creates a new endonuclease restriction site, NheI. Thisrestriction site is not present in the structural gene of SHV-1, SHV-6,SHV-8, or SHV-11, but these enzymes also have a pI of 7.6. Therefore,RFLP analysis using NheI can help distinguish between these two groupsof enzymes (Nüesch-Inderbinen et al., Eur. J. Clin. Microbiol. Infect.Dis., 39:185-191 (1996)). Isoelectric focusing data suggested that theidentity of the pI 7.6 beta-lactamase could be SHV-2, SHV-6, SHV-8 or ahyperproducer of SHV-1. To help distinguish between SHV-2 and SHV-6,SHV-8 or a hyperproducer of SHV-1, RFLP analysis on SHV-specific PCRproducts from K. pneumoniae 225 were performed using NheI. The presenceof the NheI site in the SHV-specific PCR product will result in 2 bands:219 bp and 164 bp. The absence of the NheI site will result in nocleavage and a full length fragment: 383 bp. SHV-specific PCR productsamplified from template prepared from K. pneumoniae 225 show both fulllength and cleaved products. These data suggest that SHV-1, SHV-6, orSHV-8 as well as an SHV ESBL is encoded by K. pneumoniae 225 DNA.

EXAMPLE 2 Beta-lactamases Responsible for Resistance toExpanded-Spectrum Cephalosporins among Klebsiella pneumoniae,Escherichia coli and Proteus mirabilis Isolates Recovered in SouthAfrica

Materials and Methods

Bacterial Strains

During a period of three months in 1993, 37 strains of Klebsiellapneumoniae (13 blood, 5 burn, 7 wound, 11 tracheal isolates), 4 strainsof Proteus mirabilis (all wound isolates) and 4 strains of Escherichiacoli (1 blood, 1 burn, 2 wound isolates) were collected from patients atthe following medical centers in South Africa: Tygerberg Hospital nearCape Town, King Edward VIII Hospital in Durban, Chris Hani BaragwanathHospital in Soweto and Pretoria Academic Hospital in Pretoria. Thestrains were provided in response to a request for all strains ofEnterobacteriaceae, lacking inducible beta actamases, that wereintermediate or resistant to cefotaxime or ceftazidime. The total numberof strains screened is unknown, and at this time the referring hospitalsdid not perform more sensitive screening tests for ESBL detection.Therefore accurate prevalence data were not obtained.

Thirty-four of the 43 patients involved (including all from whom bloodisolates were obtained) had received a third generation cephalosporinduring the four weeks prior to isolation of the above organisms. Fifteenpatients (including 8 blood isolate patients) were receiving eithercefotaxime or ceftazidime at the time the isolates were cultured andwere considered not to be responding to these agents.

Susceptibility Testing and Antibiotics

Antibiotic susceptibility was determined by standard disk diffusion(NCCLS Standard M2-T4, 1994) and agar dilution (NCCLS Standard M7-T2,1994) procedures. Standard powders of antimicrobial agents were kindlyprovided by the following companies: piperacillin and tazobactam(Lederle Laboratories, Wayne, N.J.); cefoxitin and imipenem, (Merck,Rathway, N.J.); cefotaxime, (Hoechst-Roussel Pharmaceuticals Inc.,Somerville, N.J.); ceftazidime, (Glaxo Group Research Ltd., Greenford,England); aztreonam and cefepime, (Bristol-Myers Squibb, Princeton,N.J.). Disks for agar diffusion were obtained from Becton DickinsonMicrobiology Systems (Cockeysville, Md.). For quality control purposes,the following quality control strains were run simultaneously with thetest organisms E. coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, E.coli ATCC 35218, and Staphylococcus aureus ATCC 29213. Throughout thisstudy, results were interpreted using NCCLS criteria for disk diffusion(NCCLS Standard M2-T4, 1994) and broth dilution (NCCLS Standard M7-T2,1994).

Double-Disk Test

All the strains were screened for the production of extended-spectrumbeta-lactamases by using the double-disk test as described by Jarlier etal., Rev. Infect. Dis. 10:867-878 (1988). A potentiation of the zones ofcefotaxime, ceftriaxone, ceftazidime or aztreonarn by clavulanic acidrepresented a positive test and was indicative of possible presence ofan extended-spectrum beta-lactamase.

Beta-lactamase Characterization

Overnight cultures in 5 ml trypticase soy broth were diluted with 45 mlfresh broth and incubated with shaking for 4 hours at 37° C. Cells wereharvested by centrifugation at 4° C., washed with 1 Mpotassium-phosphate buffer (pH 7.0), suspended and sonicated. Aftersonication, crude extracts were obtained by centrifugation at 5,858×gfor 1 hour. One strain, K. pneumoniae Pit 68, with a suspected AmpCbeta-lactamase, was induced with cefoxitin as described below in Example3. The rate of hydrolysis of 100 μM solutions of nitrocephin,cephalothin, cefotaxime, ceftazidime and aztreonam was performed byspectrophotometric assays on crude beta-lactamase extracts (Naumovski etal., Antimicrob. Agents Chemother., 36:1991-1996 (1992)).

The beta-lactamases in the sonic extracts were assessed for isoelectricpoints (pIs), general substrate and inhibitor characteristics inpolyacrylamide gels. As controls, crude beta-lactamase preparations fromthe following organisms possessing different TEM and SHV enzymes wereexamined simultaneously with the K. pneumoniae, E. coli and P. mirabilisstrains: TEM-1 [from E. coli RTEM (R6K)], TEM-2 [from E. coli 1752E(RP1)], TEM-10 [from E. coli C600 (pK2)], TEM-26 [from E. coli HB101(PJPQ101), SHV-1 [from E. coli J53 (R1010)], SHV-2 [from Klebsiellaozaenae 2180], SHV-3 [from E. coli J53 (pUD18)], SHV-4 [from E. coilJ53-2 (pUD21)] and SHV-5 [from E. coli ClaNal (pAFF2)].

DNA Amplification Using Polymerase Chain Reaction (PCR)

The organisms were inoculated into 5 ml of Luria Bertani (LB) broth(Difco, Detroit, Mich.) and incubated for 20 hours at 37° C. withshaking. Cells from 1.5 ml of overnight culture were harvested bycentrifugation at 17,310×g in an Hermle centrifuge for 5 minutes. Afterthe supernatant was decanted, the pellet was resuspended in 500 μl ofdistilled water. The cells were lysed by heating at 95° C. for 10minutes and cellular debris was removed by centrifugation for 5 minutesat 17,310×g. The supernatant was used as source of template foramplification.

The following oligonucleotide primers specific for the SHV and TEM geneswere designed by using MacVector version 4.5 (Kodak/IBI): SHV genes: A[5′-(CACTCAAGGATGTATTGTG) -3′] (SEQ ID NO:10) and B[5′-(TTAGCGTTGCCAGTGCTCG)-3′] (SEQ ID NO:11) corresponding to nucleotidenumbers 103 to 121 and 988 to 970, respectively, of Mercier et al.,Antibicrob. Agents Chemother. 34:1577-1583 (1990)); TEM genes: C[5′-(TCGGGGAAATGTGCGCG)-3′ (SEQ ID NO:5) and D[5′-(TGCTTAATCAGTGAGGCACC)-3′ (SEQ ID NO:1) corresponding to nucleotidenumbers 90 to 105 and 1062 to 1042, respectively, of Sutcliff et al.,Proc. Nat. Aca. Sci., USA, 75:3737-3741 (1978). Primers A and Bamplified a 885 base pair fragment while primers C and D amplified a 971base pair fragment. The specificity of the SHV and TEM primers foramplification of SHV and TEM genes respectively was tested by using thefollowing beta-lactamase controls; TEM-1 (pACYC177), MIR-1 (from K.pneumoniae 96D) and SHV-7 (pCLL3410).

PCR amplifications were carried out on a DNA Thermal Cycler 480instrument (Perkin-Elmer, Cetus, Norwalk, Conn.) using the Gene Amp DNAamplification kit containing AmpliTaq polymerase (Perkin Elmer, RocheMolecular Systems, Inc., Branchburg, N.J.). The composition of thereaction mixture was as follows: 10 mM Tris-HCl (pH 8.3), 50 mM KCl,0.1% TRITON X-100, 1.5 mM MgCl₂, 0.2 mM (each) of the fourdeoxynucleoside triphosphates, and 1.2 U of AmpliTaq in a total volumeof 49 μl. A total of 1 μl of sample lysate was added to the reactionmixture, and was centrifuged briefly before 50 μl of mineral oil waslayered on the surface. The PCR program consisted of an initialdenaturation step at 96° C. for 15 seconds; followed by 24 cycles of DNAdenaturation at 96° C. for 15 seconds, primer annealing at 50° C. for 15seconds and primer extension at 72° C. for 2 minutes. After the lastcycle the products were stored at 4° C. The PCR products (1/10 volume)were analyzed by electrophoresis using 1.4% agarose gels in TAE buffer(0.04 M Tris-acetate, 0.002 M EDTA [pH 8.5]). The gels were stained withethidium bromide and the PCR products were visualized with ultra-violetlight. A single band was observed for TEM amplified products using asingle primer set. Two amplified products were observed with the SHVprimer set. The larger product which corresponded to the expected sizeof the SHV specific product was gel purified using a 1.4% agarose in TAEgel and the purified PCR product was used for sequence analysis.

PCR products were sequenced by automated PCR cycle-sequencing withdye-terminator chemistry using a DNA stretch sequencer from AppliedBiosystems.

Results

Resistance Phenotypes

All the strains except K. pneumoniae Pit 68, gave a positive diskpotentiation when using cefotaxime, ceftriaxone, aztreonam and/orceftazidime disks. Minimum inhibitory concentrations of piperacillin,piperacillin/tazobactam, cefotaxime, ceftazidime, aztreonam andcefoxitin revealed 3 different resistance phenotypes (Kpn1, 2 and 3) inthe K. pneumoniae strains, and 2 (Ecl and 2) in E. coli strains (Table4).

TABLE 4 Minimum Inhibitory Concentrations of the Different ResistancePhenotypes Observed in K. pneumoniae, E. coli and P. mirabilis MIC range(μg/ml)^(a) Resistance phenotype No. strains pip tzp ctx caz atm fox fepimi K. pneumoniae Kpn1 8 >128 2-4 0.25-1 >128 16-64 2-4 0.12 0.12 Kpn228 >128 2—>128 4-64 4-128 1-128 2-8 0.5-4 0.12-1 Kpn3 1 64 16 4 4 2 >1280.12 0.12 E. coli Ec1 3 >128 1 1 >128 16 4 0.12 0.12 Ec2 1 >128 32 16 42 8 2 0.12 P. mirabilis 4 128 0.25-0.5 0.25-0.5 16-64 0.5-2 2-4 2 0.5-1^(a)pip—piperacillin, tzp—piperacillin/tazobactam (4 μg/ml),ctx—cefotaxime, caz—ceftazidime, atm—aztreonam, fox—cefoxitin,fep—cefepime, imi—imipenem.

The phenotypes Kpn1 and Ec1 involved high level resistance toceftazidime (MIC>128 μg/ml) but susceptibility to cefotaxime (MIC range0.25-1 μg/ml), while Kpn2 and Ec2 involved decreased susceptibility toboth cefotaxine (MIC range 4-64 μg/ml) and ceftazidime (MIC range 4-128μg/ml). Kpn 3, represented by K. pneumoniae Pit 68, involved resistanceto cefoxitin (MIC>128 μg/ml) and decreased susceptibility to cefotaxime,ceftazidime and aztreonam (MIC>2 μg/ml) (Table 4). The P. mirabilisisolates showed decreased susceptibility to ceftazidime (MIC range 16-64μg/ml) and susceptibility to cefotaxime (MIC range 0.25-0.5 μg/ml).

Beta-lactamases

Strains representing the Kpnl and Ecl phenotypes producedbeta-lactamases with pI values of 5.6 and 7.6 respectively, whilephenotypes Kpn2 and Ec2 involved enzymes with pI's of 5.4, 7.6, and 8.2(Table 5). K. pneumoniae Pit 68, representing phenotype Kpn3, producedtwo beta-lactamases with pls of 5.4 and 8.0. The P. mirabilis strainsshowed a single enzyme with a pI value of 5.6 (Table 5). The enzymes ofpI 5.4, 5.6, 7.6 and 8.2 aligned with TEM-1 (pI 5.4), TEM-10 or 26 (pI5.57), SHV-1, 2 or 8 (pI 7.6) and SHV-5 (pI 8.2) respectively (Table 5).It was therefore necessary to investigate these enzymes further. Onisoelectric focusing gels, all of the beta-lactamases except for theenzymes with a pI of 8.0 were inhibited by clavulanate, a characteristicof Bush group 2 enzymes. The enzyme with a pI of 8.0 was inhibited bycloxacillin which correlates with Bush group 1 cephalosporinases. Thesubstrate-based technique showed hydrolysis of 0.75 Fg/ml cefotaxime atthe bands focusing at: 5.6, 8.0, 8.2 and some enzymes with a pI of 7.6(Table 5). Control enzymes of TEM-10, TEM-26, SHV-2 and SHV-5 showedhydrolysis of cefotaxime in this assay (Table 5).

TABLE 5 Characteristics of Beta-lactamases Produced by DifferentResistance Phenotypes ctx Inhibited^(b) Resistance No. hydro- by: Mostsimilar phenotype strains pI lysis^(a) clox clav Beta-lactamases K.pneumoniae Kpn1 8 5.6 Yes No Yes TEM-10 or 26 7.6 No No Yes SHV-1 Kpn228 5.4 No No Yes TEM-1 7.6 Yes No Yes SHV-2 or 8 8.2 Yes Yes No SHV-5Kpn3 1 5.4 No No Yes TEM-1 8.0 Yes Yes No AmpC E. coli Ec1 3 5.4 No NoYes TEM-1 5.6 Yes No Yes TEM-10 or 26 Ec2 1 5.4 No No Yes TEM-1 7.6 YesNo Yes SHV-2 or 8 P. mirabilis 4 5.6 Yes No Yes TEM-10 or 26^(a)Hydrolysis of 0.75 μg/ml cefotaxime (ctx) used in substrate-basedisoelectric focusing overlay technique (Hibbert-Rodgers et al., J.Antimicrob. Chemother., 33:707-720 (1994)). ^(b)Inhibitors used inisoelectric focusing overlay technique were clav, clavulanic acid; clox,cloxacillin (Huletsky et al., Antimicrob. Agents and Chemother.,34:1725-1732 (1990)).

Hydrolysis assays with nitrocefin, cefotaxime, ceftazidime and aztreonamwere performed on strains possessing single beta-lactamases. All thestrains assayed hydrolyzed cefotaxime, ceftazidime and aztreonam to someextent (Table 6).

TABLE 6 Hydrolysis Profiles of Cell Extracts Containing a SingleBeta-lactamase Hydrolysis (nmol of substrate^(a) hydrolyzed/min/mg ofprotein) Beta- lactamase Nitro- Cefo- Cef- Strain (pI) cefin taximetazidime Aztreonam K. pneumoniae Pit 16 5.6 114 4 3 0.6 Pit 100 7.6 15911  0.1 0.9 Pit 82 8.2 136 11  0.2 0.9 E. coli Pit 64 5.6 275 1 2 0.4Pit 56 7.6 143 9 0.1 0.8 P. mirabilis Pit 85 5.6 138 5 1 0.5 ^(a)100 μMsolution of substrate

DNA Ampilification and Sequencing

The DNA from organisms producing single beta-lactamases were amplifiedand sequenced. Strains producing ESBLs with pIs of 5.6, which alignedwith TEM-10 and TEM-26, were amplified with the TEM primers (Table 7).Amino acids at positions 104, 164 and 240 (Ambler numbering (1)) wereutilized to determine that this enzyme was more similar to TEM-26. Aminoacids deduced from amplicon sequences included lysine at position 104,serine at position 164 and glutamine at position 240 (Table 7). Strainsproducing ESBLs with pI values 7.6 and 8.2, which aligned with SHV-2 andSHV-5 respectively, were amplified with SHV primers (Table 7). Aminoacids at positions 205, 238 and 240 (Labia numbering (2)) were used toidentify the ESBL involved. Arginine at position 205, serine at position238 and glutamic acid at position 240 of the deduced amino acid sequenceof strains producing an ESBL with a pI of 7.6 indicated the presence ofSHV-2 (Table 7). K. pneumoniae Pit 82, producing an ESBL with a pI 8.2,had a lysine at position 240 indicating the presence of SHV-5 (Table 7).

TABLE 7 Identification of Extended-Spectrum Beta-lactamases Occurring inSouth Africa TEM^(b) SHV^(c) Amplification with aa aa aa aa Aa aaStrain^(a) pI TEM primers SHV primers 104 164 240 205 238 240beta-lactamase K. pneumoniae Pit 16 5.6 Yes No Lys Ser Glu — — —TEM-26-type Pit 100 7.6 No Yes — — — Arg Ser Glu SHV-2 Pit 82 8.2 No Yes— — — Arg Ser Lys SHV-5 E. coli Pit 64 5.6 Yes No Lys Ser Glu — — —TEM-26-type Pit 56 7.6 No Yes — — — Arg Ser Glu SHV-2 P. mirabilis Pit85 5.6 Yes No Lys Ser Glu — — — TEM-26-type ^(a)Strains with singlebeta-lactamases used for sequencing ^(b)Numbering according to Sutcliffeet al., Proc. Nat. Aca. Sci., USA, 75, 3737-3741 (1978). ^(c)Numberingaccording to Mercier et al., Antimicrob. Agents Chemother., 34,1577-1583 (1990).

EXAMPLE 3 Plasmid-Mediated Resistance to Expanded-SpectrumCephalosporins among Enterobacter aerogenes

Materials and Methods

Bacterial Strains

Among all E. aerogenes recovered from clinical specimens during aneighteen month period (September 1993 to March 1995), thirty-one E.aerogenes strains showing a resistance phenotype different from thatobserved with derepressed mutants normally encountered at the HunterHolmes McGuire Medical Center, Richmond, Virginia were selected for thisstudy. The strains selected were intermediate to ceftriaxone butresistant to ceftazidime when tested with the Vitek automatedsusceptibility system (bioMerieux Vitek, St. Louis, Mo.). Derepressedmutants previously isolated from this center were usually resistant toboth ceftriaxone and ceftazidime.

Susceptibility Testing

Antibiotic susceptibilities were determined by standard disk-diffusion(NCCLS Standard M2-A6) and agar-dilution (NCCLS Standard M7-A4)procedures. Disks were obtained from Becton Dickinson MicrobiologySystems (Cockeysville, Md.). Disk-diffusion susceptibilities to thefollowing antibiotics were determined, ampicillin; amoxicillinclavulanic acid; aztreonam; cefazolin; cefoxitin; cefuroxime;cefotaxime; ceftriaxone; ceftazidime; cefepime; imipenem; gentamicin;trimethoprim/sulfamethoxazole and ciprofloxacin. Standard powders ofantimicrobial agents for minimum inhibitory concentration (MICs) werekindly provided by the following companies: cefoxitin and imipenem,(Merck, Rathway N.J.); cefotaxime, (Hoechst-Roussel PharmaceuticalsInc., Somerville, N.J.); ceftazidime, (Glaxo Group Research Ltd.,Greenford England); aztreonam and cefepime, (Bristol-Myers Squibb,Princetown, N.J.) and gentamicin, (Schering-Plough, Liberty Comer,N.J.). The following quality control strains were run simultaneouslywith the test organisms Escherichia coli ATCC 25922, Pseudomonasaeruginosa ATCC 27853, and E. coli ATCC 35218. Throughout this study,results were interpreted using NCCLS criteria for disk diffusion (NCCLSM2-A6) and broth dilution (NCCLS M7-A4).

Double-Disk Potentiation Test

This test described by Jarlier et al., Rev. Infect. Dis. 10:867-878(1988), using ceftazidime, cefotaxone, cefotaxime and aztreonam diskswas performed on the strains to screen for possible ESBL production.This test is a modification of the disk diffusion susceptibility test inthat cefotaxime, ceftriaxone, ceftazidime and aztreonam disks are placed30 mm from disks containing amoxycillin/clavulanic acid. A potentiationof the zones of cefotaxime, ceftriaxone, ceftazidime or aztreonam byclavulanic acid represented a positive test and was indicative ofpossible ESBL production.

Beta-lactamase Preparation Isoelectric Focusing and Assays

Overnight cultures in 5 mls Mueller-Hinton broth were diluted with 95 mlfresh broth and incubated with shaking for 90 minutes at 37° C.Cefoxitin, at a concentration of ¼ of the MIC, was added for inductionwhile sterile medium was used in the non-induced cultures and incubatedfor an additional 2 hours. The induction process was stopped by adding 1mM 8-hydroxyquinoline solution to each culture. Cells were harvested bycentrifugation at 4° C., washed with 1M potassium-phosphate buffer (pH7.0), suspended and sonicated. After sonication, crude extracts wereobtained by centrifugation at 6,000 rpm for 1 hour. The beta-lactamasesin the sonic extracts were assessed for isoelectric points (pIs), andsubstrate and inhibitor profiles in polyacrylamide gels. The rates ofhydrolysis of cephalothin were determined by ultra-violetspectrophotometric assay (O'Callghan et al., Antimicrob. Agents andChemother., 1966, 337-343 (1967)). As controls, crude beta-lactamasepreparations from the following organisms possessing differentSHVenzymes were evaluated simultaneously with those obtained from theEnterobacter strains: SHV-1 [from E. coli J53(R1010)], SHV-2 (fromKlebsiella ozaenae 2180), SHV-3 [from E. coli J53-2(pUD18)], SHV-4 [fromE. coli J53-2(pUD21)] and SHV-5 [from E. coli Cila Nal (pAFF2)].

Isolation of Plasmids

The organisms were inoculated into 5 ml of LB (Luria Bertani) broth[Difco (Detroit, Mich.)] and incubated for 20 hours at 37° C. withshaking. Cells from 1.5 ml of overnight culture were harvested bycentrifugation in an Eppendorf centrifuge for 5 minutes. After thesupernatant was decanted, the pellet was resuspended in TRITON X100 1%in TE buffer for 10 minutes. Plasmid DNA was then isolated by thealkaline extraction method of Bimboim et al., Nucleic Acids Res., 7:1513(1979), and separated by electrophoresis in 0.8% agarose gel (Sigma, St.Louis, Mo.) in TAE buffer (0.04M Tris-acetate, 0.002M EDTA [pH 8.5]).The gel was stained with ethidium bromide and plasmid bands werevisualized using ultra-violet light.

Conjugation Experiments

To determine if the resistance was transferable, transconjugationexperiments were performed using Escherichia coli C60ON(Nal^(r)) asrecipient (Maniatis et al., Molecular Cloning: A Laboratorv Manual, ColdSpring Harbor, N.Y., 1982). The filter paper mating technique withovernight incubation at 37° C. were performed as described previously(Philippon et al., Antimicrob. Agents Chemother., 33:1131-1136 (1989)).Transconjugants were selected on LB (Luria Bertani) agar [Difco(Detroit, Mich.)] plates containing 12 μg per ml nalidixic acid and 20μg per ml ampicillin.

DNA Amplification Using the Polymerase Chain Reaction (PCR)

Organisms were inoculated into 5 ml of LB (Luria Bertani) broth [Difco(Detroit, Mich.)] and incubated for 20 hours at 37° C. with shaking.Cells from 1.5 ml of overnight culture were harvested by centrifugationat 13,000 rpm in an Eppendorf centrifuge for 5 minutes. After thesupernatant was decanted, the pellet was resuspended in 500 μl ofsterile deionized water. The cells were lysed by heating to 95° C. for10 minutes and cellular debris were removed by centrifugation for 5minutes at 13,000 rpm. The supernatant was used as a source of templatefor amplification. Oligonucleotide primers specific for SHV genes wereselected from a consensus alignment sequence generated by the MacVector4.5 (Kodak/IBI) software package from the published nucleic acidsequence of SHV-1 (Mercier et al., Antimicrob. Agents Chemother.,34:1577-1583 (1990)), SHV-2 (Jacoby et al., Antimicrob. AgentsChemother., 35:1697-1704 (1991)), SHV-5 (Billot-Klein et al.,Antimicrob. Agents Chemother., 34:2439-2441 (1990)) and SHV-7 (Bradfordet al., Antimicrob. Agents Chemother., 39:899-905 (1995)). The PCRprimers used were A [5′-(CACTCAAGGATGTATTGTG)-3′] (SEQ ID NO:10) and B[5′-(TTAGCGTTGCCAGTGCTCG)-3′] (SEQ ID NO:11) which amplified a 781 basepair fragment. Primer specificity controls included the TEM-1, MIR-1 andSHV-7 beta-lactamase genes. PCR amplifications were carried out on a DNAThermal Cycler 480 instrument (Perkin-Elmer, Cetus, Norwalk, Conn.)using the GeneAmp” DNA amplification kit containing AmpliTaq” polymerase(Perkin Elmer, Roche Molecular Systems, Inc., Branchburg, N.J.). Thecomposition of the reaction mixture was as follows: 10 mM Tris-HCl (pH8.3), 50 mM KCl, 0.1% TRITON X-100, 1.5 mM MgCl₂, 0.2 mM (each) of thefour deoxynucleoside triphosphates, and 1.2 U of AmpliTaq” in a totalvolume of 49 μl. A total of 1 μl of sample lysate was added to thereaction mixture, and was centrifuged briefly before 50 μl of mineraloil was layered on the surface. The PCR program consisted of an initialdenaturation step at 96° C. for 30 seconds; followed by 24 cycles of DNAdenaturation at 96° C. for 30 seconds, primer annealing at 50° C. for 15seconds and primer extension at 72° C. for 2 minutes. After the lastcycle the products were stored at 4° C. The PCR products were analyzedby electrophoresis using 1.4 % agarose gels in TAE buffer. The gels werestained with ethidium bromide and the PCR products were visualized withultra-violet light.

Results

Bacterial Strains

Twenty-four of the 31 strains from Hunter Holmes McGuire Medical Centeroriginated from patients in 2 spinal cord injury wards (SCW1, SCW2)while 3 strains were isolated from patients in the medical intensivecare unit, 3 isolates were recovered from patients attending thesurgical outpatient clinic, and 1 isolate was recovered from a patientin a general surgery ward. Disk diffusion susceptibility tests showedall the strains to be resistant to ampicillin, amoxycillin-clavulanate,cefazolin, cefuroxime, trimethoprim/sulfamethoxazole and susceptible tociprofloxacin. MICs for cefoxitin, cefotaxime, ceftazidime, aztreonam,cefepime, imipenem and gentamicin are summarized in Table 8, below. Allstrains were susceptible to cefepime and imipenem but showed decreasedsusceptibility to cefotaxime, ceftazidime and aztreonam. MICs forgentamicin ranged from 8 μg/ml to >128 μg/ml for 25 of 37 (67%)isolates. All the strains selected for this study showed a positivedouble disk test when using cefotaxime and ceftriaxone disks.

TABLE 8 Minimum Inhibitory Concentrations and Characteristics ofBeta-lactamases Produced by E. aerogenes Enzyme Characteristics No. ofBeta-lactamase^(b) Ctx Inhibited by^(d): MIC (μg/ml) range^(a) StrainsPresent pI Hydrolysis^(c) clox clav Inducible^(e) fox ctx caz atm fepimi gm 1 Bush group 1 8.3 no yes no yes >256 1 4 1 0.12 0.5 8 Bush group2be 6.9 yes no yes no (SHV-3) 29 Bush group 1 8.3 no yes no yes >256 1-28-32 32-64 0.12-1 0.5 1-16 Bush group 2be 7.8 yes no yes no (SHV-4) 1Bush group 1 8.3 no yes no yes >256 1-2 32 64 1 1 >128 Bush group 2be8.0 yes no yes no (SHV-5) ^(a)Cefoxitin (fox); cefotaxime (ctx);ceftazidime (caz); aztreonam (atm); cefepime (fep); imipenem (imi);gentamicin (gm). ^(b)Based on Bush-Jacoby-Medeiros classification(Antimicrob. Agents Chemother., 39:899-905 (1995)). Beta-lactamaselisted in parentheses is the one most similar to the group 2be enzymeproduced by the Enterobacter strains. ^(c)Hydrolysis of 0.75 μg/mlcefotaxime (ctx) used in substrate-based isoelectric focusing overlaytechnique (Bauernfeind et al., Infection, 18:294-298 (1990)).^(d)Inhibitors used in isoelectric focusing overlay technique were clav,clavulanic acid; clox, cloxacillin (Sanders et al., Clin. Microbiol.Rev., 10:220-241 (1997)). ^(e)Inducible by cefoxitin.

Characteristics of Beta-lactamases.

All the Enterobacter isolates possessed a Bush group 1 induciblebeta-lactamase with an alkaline pI of 8.3 which was sensitive toinhibition by cloxacillin but not clavulanic acid (Table 8, above).Additional Bush group 2be enzymes with pIs resembling SHVbeta-lactamases were also present in all the strains (Table 8). Threedifferent Bush group 2be enzymes were detected in the species ofEnterobacter (Table 8): the majority of isolates (29 of 31) produced anenzyme with a pI of 7.8 which aligned with SHV-4. One isolate producedan enzyme with a pI of 6.8 which aligned with SHV-3, and one isolateproduced an enzyme with a pI of 8.2 which aligned with SHV-5.

Plasmid Profiles

A variety of different plasmids with sizes ranging from 10 kb toapproximately 60 kb were visualized with electrophoresis (Table 9,below). Furthermore, eight different plasmid patterns were observed withthe number of plasmids ranging from 0 to 5 per organism (Table 9). Noplasmids were visualized in 3 strains, which included the strain whichproduced an enzyme resembling SHV-5 (Table 9). Three differentsusceptibility profiles were identified (Table 9). The majority oforganisms isolated were resistant to ceftazidime, aztreonam,trimethoprim/sulfamethoxazole and gentamicin. This antibiogram wasassociated with the production of β-lactamases resembling SHV-4 andSHV-5 and were isolated from the spinal cord injury wards 1 and 2,medical intensive care unit, general surgical ward as well as theoutpatient clinic (Table 9). Eight of thirty strains showing 3 differentplasmid profiles (a, b and f) producing an enzyme resembling SHV-4isolated from SCW1 as well as the surgical outpatient clinic weresusceptible to gentamicin while the E. aerogenes strain producing anenzyme resembling SHV-3 appeared susceptible to ceftazidime andaztreonam (Table 9). Seven different plasmid profiles (b-h) wereobserved among E. aerogenes isolated from the spinal cord injury wardSCW1 while only 4 patterns (c, d, g and h) were observed among thoserecovered from SCW2 (Table 9). Plasmid profile b, observed in 6isolates, consisting of 5 plasmids ranging from 50 kb to 10 kb andplasmid profile f, observed in 1 isolate (3 plasmids ranging from 60 kbto 10 kb) were unique to SCW1 (Table 9). Two of the three strainsisolated from MICU possessed 4 plasmids ranging from 60 kb to 10 kb(plasmid profile c) while no plasmids were visualized from the otherstrain (plasmid profile h) [Table 9]. These organisms produced an enzymeresembling SHV-4 and were resistant to ceftazidime, aztreonam,trimethoprim/sulfamethoxazole and gentamicin. The E. aerogenes strainsoriginating from the surgical outpatient clinic had 2 differentantibiograms and plasmid profiles. Two of three isolates, possessingplasmid profile e, were resistant to ceftazidime, aztreonam,trimethoprim/sulfamethoxazole and gentamicin while the remaining isolatewith plasmid profile a, appeared susceptible to gentamicin (Table 9).All these organisms produced an enzyme resembling SHV-4.

TABLE 9 Plasmid Profiles of Enterobacter aerogenes Approximate MostLikely Plasmid profile No. plasmids Size (kilobases) Ward (no. ofisolates)^(a) Antibiogram^(b) ESBL A 5 50, 35, 20, 15, 12 OPC(1)CAZ/ATM/SXT SHV-4 B 5 50, 45, 35, 20, 10 SCW1(6) CAZ/ATM/SXT SHV-4 C 460, 45, 20, 10 SCW1(5) CAZ/ATM/SXT/G SHV-4 SCW2(2) CAZ/ATM/SXT/G SHV-4MICU(2) CAZ/ATM/SXT/G SHV-4 D 4 45, 35, 20, 10 SCW1(2) CAZ/ATM/SXT/GSHV-4 SCW2(1) CAZ/ATM/SXT/G SHV-4 E 3 60, 50, 14 SCW1(2) CAZ/ATM/SXT/GSHV-4 OPC(2) CAZ/ATM/SXT/G SHV-4 F 3 60, 50, 10 SCW1(1) CAZ/ATM/SXTSHV-4 G 2 50, 10 SCW1(1) SXT/G SHV-3 SCW2(2) CAZ/ATM/SXT/G SHV-4 SGW(1)CAZ/ATM/SXT/G SHV-4 H 0 — SCW1(1) CAZ/ATM/SXT/G SHV-5 SCW2(1)CAZ/ATM/SXT/G SHV-4 MICU(1) CAZ/ATM/SXT/G SHV-4 ^(a)OPC; outpatientclinic, SCW1; spinal cord injury ward 1, SCW2; spinal cord injury ward2, MICU; medical intensive care unit, SGW; surgical general ward.^(b)Resistance to CAZ; ceftazidime, ATM; aztreonam, SXT;trimethoprim/sulfamethoxazole, G; gentamicin. Conjugation Experiments.

The following strains were selected for conjugation with E. coli C600N.E. aerogenes 187 producing an enzyme with a pI of 6.8 resembling SHV-3;E. aerogenes 200 and E. aerogenes 220 producing enzymes with pIs of 7.8resembling SHV-4; and E. aerogenes 184 producing an enzyme with a pI of8.2 resembling SHV-5. All the strains also possessed an inducible Bushgroup 1 beta-lactamase with a pI of 8.3. A plasmid of approximately 50kb was transferred from E. aerogenes 187, E. aerogenes 200 and E.aerogenes 220 to E. coli C600N (Table 10, below). No plasmids werevisualized in E. aerogenes 184 or its transconjugant E. coli JPO4/tr(Table 10). Isoelectric focusing performed on the Enterobacter strainsand their respective transconjugants showed the beta-lactamasesresembling SHV-3, SHV-4 and SHV-5 present in both donors and recipients.The transfer of plasmids encoding SHV beta-lactamase genes into E. coliC60ON was accompanied by resistance to gentamicin andtrimethroprim/sulfamethoxazole and decreased susceptibility tocefotaxime, ceftazidime and aztreonam (Table 10).

TABLE 10 Characteristics of Enterobacter Strains and RespectiveTransconjugants Plasmids Beta- Most likely (approximate size, lactamasesBeta- Strains^(a) kilobases) (pI) lactamase E. coli C600N — — — E.aerogenes 187 50, 10 8.3, 6.8 AmpC, SHV-3 E. coli JP01/tr 50 6.8 SHV-3E. aerogenes 200 60, 50, 14 8.3, 7.8 AmpC, SHV-4 E. coli JP02/tr 50, 107.8 SHV-4 E. aerogenes 220 60, 50, 10 8.3, 7.8 AmpC, SHV-4 E. coliJP03/tr 50 7.8 SHV-4 E. aerogenes 184 — 8.3, 8.2 AmpC, SHV-5 E. coliJP04/tr — 8.2 SHV-5 ^(a) E. aerogenes 187, 200, 220 and 184 were donors;E. coli C600N served as recipient and JP01, 2, 3 and 4 were therespective transconjugants

DNA Amplification

The strains used in the conjugation experiments were selected foramplification with PCR; E. aerogenes 187 (pI 6.8), E. aerogenes 200 (pI7.8), E. aerogenes 220 (pI 7.8),E. aerogenes 184 (pI of 8.2) as well astheir respective transconjugants E. coli JP01/tr (pI 6.8), E. coliJP02/tr (pI 7.8), E. coli JP03/tr (pI 7.8) JP04/tr (pI 8.2). Controlstrains producing SHV-3, SHV-4, SHV-5 and SHV-7 were used as positivecontrols while E. coli C60ON was used as a negative control. A 781 basepair fragment specific for SHV beta-lactamases was amplified in E.aerogenes 187, E. aerogenes 200, E. aerogenes 220, E. aerogenes 184 andtheir respective transconjugants as well as the positive controls (Table10, above). No amplification was observed with E. coli C600N (Table 10).Therefore, the ESBLs produced by these strains are indeed derivatives ofan SHV beta-lactamase.

The complete disclosures of the patents, patent documents, andpublications cited herein are incorporated by reference in theirentirety as if each were individually incorporated. Variousmodifications and alterations to this invention will become apparent tothose skilled in the art without departing from the scope and spirit ofthis invention. It should be understood that this invention is notintended to be unduly limited by the illustrative embodiments andexamples set forth herein and that such examples and embodiments arepresented by way of example only with the scope of the inventionintended to be limited only by the claims set forth herein as follows.

Sequence Listing Free Text

SEQ ID NO:1-45 Primer

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 45 <210> SEQ ID NO: 1 <211>LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Primer <400> SEQUENCE: 1 tgcttaatca gtgaggcacc 20 <210> SEQ ID NO: 2<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Primer <400> SEQUENCE: 2 agatcagttg ggtgcacgag 20 <210> SEQ IDNO: 3 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: Primer <400> SEQUENCE: 3 cttggtctga cagttacc 18<210> SEQ ID NO: 4 <211> LENGTH: 15 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Primer <400> SEQUENCE: 4 tgtcgccctt attcc 15<210> SEQ ID NO: 5 <211> LENGTH: 15 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Primer <400> SEQUENCE: 5 tcggggaaat gtgcg 15<210> SEQ ID NO: 6 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Primer <400> SEQUENCE: 6 atcgtccacc atccactgca20 <210> SEQ ID NO: 7 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Primer <400> SEQUENCE: 7 gggaaacgga actgaatgag20 <210> SEQ ID NO: 8 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Primer <400> SEQUENCE: 8 tagtggatct ttcgctccag20 <210> SEQ ID NO: 9 <211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Primer <400> SEQUENCE: 9 gctctgcttt gttattc 17<210> SEQ ID NO: 10 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Primer <400> SEQUENCE: 10 cactcaagga tgtattgtg19 <210> SEQ ID NO: 11 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Primer <400> SEQUENCE: 11 ttagcgttgc cagtgctcg19 <210> SEQ ID NO: 12 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Primer <400> SEQUENCE: 12 ggaacagact gggctttcatc 21 <210> SEQ ID NO: 13 <211> LENGTH: 15 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Primer <400> SEQUENCE: 13 ggacatccccttgac 15 <210> SEQ ID NO: 14 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Primer <400> SEQUENCE: 14 gtggattcacttctgccacg 20 <210> SEQ ID NO: 15 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Primer <400> SEQUENCE: 15 cttctggcatgccctatgag 20 <210> SEQ ID NO: 16 <211> LENGTH: 24 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Primer <400> SEQUENCE: 16 catgacccagttcgccatat cctg 24 <210> SEQ ID NO: 17 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Primer <400> SEQUENCE:17 attcgtatgc tggatctcgc cacc 24 <210> SEQ ID NO: 18 <211> LENGTH: 22<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: Primer <400>SEQUENCE: 18 ctggcaacca caatggactc cg 22 <210> SEQ ID NO: 19 <211>LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Primer <400> SEQUENCE: 19 gccagttcag catctcccag cc 22 <210> SEQ ID NO:20 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Primer <400> SEQUENCE: 20 cgtgaccaac aacgcccagc 20 <210> SEQID NO: 21 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: Primer <400> SEQUENCE: 21 ccagatagcg aatcagatcg c21 <210> SEQ ID NO: 22 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Primer <400> SEQUENCE: 22 ccagccgatg ctcaaggag19 <210> SEQ ID NO: 23 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Primer <400> SEQUENCE: 23 cacgaacgcc acataggcg19 <210> SEQ ID NO: 24 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Primer <400> SEQUENCE: 24 ggcattggga tagttgcggttg 22 <210> SEQ ID NO: 25 <211> LENGTH: 25 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Primer <400> SEQUENCE: 25 ttactacaaggtcggcgaca tgacc 25 <210> SEQ ID NO: 26 <211> LENGTH: 22 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Primer <400> SEQUENCE:26 ggatcacact attacatctc gc 22 <210> SEQ ID NO: 27 <211> LENGTH: 22<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: Primer <400>SEQUENCE: 27 cgtatggttg agtttgagtg gc 22 <210> SEQ ID NO: 28 <211>LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Primer <400> SEQUENCE: 28 gcgacctggt taactacaat ccc 23 <210> SEQ ID NO:29 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Primer <400> SEQUENCE: 29 cggtagtatt gcccttaagc c 21 <210> SEQID NO: 30 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: Primer <400> SEQUENCE: 30 cggaaaagca cgtcgatggg 20<210> SEQ ID NO: 31 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Primer <400> SEQUENCE: 31 gcgatatcgt tggtggtgcc20 <210> SEQ ID NO: 32 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Primer <400> SEQUENCE: 32 ctcgatgatg cgtgcttcgc20 <210> SEQ ID NO: 33 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Primer <400> SEQUENCE: 33 gcgactgtga tgtataaacg20 <210> SEQ ID NO: 34 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Primer <400> SEQUENCE: 34 cgtcgctcac catatctccc20 <210> SEQ ID NO: 35 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Primer <400> SEQUENCE: 35 cctctcgtgc tttagacccg20 <210> SEQ ID NO: 36 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Primer <400> SEQUENCE: 36 cgctgggaaa cctattcgg19 <210> SEQ ID NO: 37 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Primer <400> SEQUENCE: 37 ctgccatcca gtttcttcggg 21 <210> SEQ ID NO: 38 <211> LENGTH: 21 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Primer <400> SEQUENCE: 38 ggtggcattgacaaattctg g 21 <210> SEQ ID NO: 39 <211> LENGTH: 18 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Primer <400> SEQUENCE:39 cccaccatgc gacaccag 18 <210> SEQ ID NO: 40 <211> LENGTH: 19 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Primer <400> SEQUENCE:40 tgtgcaacgc aaatggcac 19 <210> SEQ ID NO: 41 <211> LENGTH: 23 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Primer <400> SEQUENCE:41 cgaccccaag tttcctgtaa gtg 23 <210> SEQ ID NO: 42 <211> LENGTH: 22<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: Primer <400>SEQUENCE: 42 aggcacgata gttgtggcag ac 22 <210> SEQ ID NO: 43 <211>LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Primer <400> SEQUENCE: 43 cactcaaccc atcctaccca cc 22 <210> SEQ ID NO:44 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Primer <400> SEQUENCE: 44 cgaacgaatc attcagcacc g 21 <210> SEQID NO: 45 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: Primer <400> SEQUENCE: 45 cggcaatgtt ttactgtagc gcc23

What is claimed is:
 1. A primer selected from the group consisting of:5′-TGC TTA ATC AGT GAG GCA CC-3′ (SEQ ID NO:1); 5′-AGA TCA GTT GGG TGCACG AG-3′ (SEQ ID NO:2); 5′-TTA GCG TTG CCA GTG CTC G-3′ (SEQ ID NO:11);5′-GGA ACA GAC TGG GCT TTC ATC-3′ (SEQ ID NO:12); 5′-GGA CAT CCC CTTGAC-3′ (SEQ ID NO:13); 5′-GTG GAT TCA CTT CTG CCA CG-3′ (SEQ ID NO:14);5′-CTT CTG GCA TGC CCT ATG AG-3′ (SEQ ID NO:15); 5′-CAT GAC CCA GTT CGCCAT ATC CTG-3′ (SEQ ID NO:16); 5′-ATT CGT ATG CTG GAT CTC GCC ACC-3′(SEQ ID NO:17); 5′-CCA GCC GAT GCT CAA GGA G-3′ (SEQ ID NO:22); 5′-CACGAA CGC CAC ATA GGC G-3′ (SEQ ID NO:23); 5′-CGA ACG AAT CAT TCA GCACCG-3′ (SEQ ID NO:44); 5′-CGG CAA TGT TTT ACT GTA GCG CC-3′ (SEQ IDNO:45); and complements thereof.
 2. A method for identifying abeta-lactamase in a clinical sample, the method comprising: providing apair of oligonucleotide primers, wherein at least one of the primers isselected from the primers of claim 1, and further wherein one primer ofthe pair is complementary to at least a portion of the beta-lactamasenucleic acid in the sense stand and the other prier of each pair iscomplementary to at least a portion of the beta-lactamase nucleic acidin the antisense strand; annealing the primers to the beta-lacmasenucleic acid; simultaneously extending the annealed primers from a 3′terminus of each primer to synthesize an extension product that iscomplementary to the nucleic acid strands annealed to each primerwhcrein each extension product after separation from te beta-lactamasenucleic acid serves as a template for the synthesis of an extensionproduct for the other primer of each pair; separating the amplifiedproducts; and analyzing the separated amplified products for a regioncharacteristic of the beta-laclamase.
 3. A diagnostic kit for detectinga beta-lactamase which comprises packaging, containing, separatelypackaged: (a) at least one primer pair capable of hybridizing tobeta-lactamasc nucleic acid of interest, wherein at least one of theprimers is selected from the primers of claim 1; (b) a positive andnegative control; and (c) a protocol for identification of thebeta-lactamase nucleic acid of interest.
 4. A method for identing abeta-lactamase in a clinical sample, the method comprising: providing apair of oligonucleotide pimers specific for nucleic acid characteristicof the AmpC beta-lactamase enzyme found in Enterobacter cloacae, whereinone primer of the pair is complementary to at least a portion of thebeta-lactamase nucleic acid in the sense strand and the other primer ofeach pair is complementary to at least a portion of the beta-lactamasenucleic acid in the antisense strand; annealing the primers to thebeta-lactamase nucleic acid; simultaneously extending the annealedprimers from a 3′ terminus of each primer to synthesize an extensionproduct that is complementary to the nucleic acid strands annealed toeach primer wherein each extension product after separation from thebeta-lactamase nucleic acid serves as a template for the synthesis of anextension product for the other primer of each pair; separaing theamplified products; and analyzing the separated amplified products for aregion characteristic of the beta-lactamase.
 5. The method of claim 4wherein the primers are selected from the group consisting of: 5′-GGAACA GAC TOG GCT TTC ATC-3′ (SEQ ID NO:12); 5′-GGA CAT CCC CTT GAC-3′(SEQ ID NO:13); 5′-GTG GAT TCA CTT CTG CCA CG-3′ (SEQ ID NO:14); 5′-CTTCTG GCA TGC CCT ATG AG-3′ (SEQ ID NO:15); 5′-CAT GAC CCA GTT CGC CAT ATCCTG-3′ (SEQ ID NO:16); 5′-ATT CGT ATG CTG GAT CTC GCC ACC-3′ (SEQ IDNO:17). 5′-CGA ACG AAT CAT TCA GCA CCG-3′ (SEQ ID NO:44); 5′-CGG CAA TGTTTT ACT GTA GCG CC-3′ (SEQ ID NO:45); and complements thereof.
 6. Adiagnostic ldt for detecting a beta-lactamase which comprises packaging,containng, separately packaged: (a) at least one primer pair capable ofhybridizing to beta-latamase nucleic acid of interest, wherein theprimers are specific for nucleic acid characteristic of the AmpCbeta-lactamase enzyme found in Enterobacter cloacae; (b) a positive andnegative control; and (c) a protocol for identification of thebeta-lactamase nucleic acid of interest.
 7. The kit of claim 5 whereinthe primers are selected from the group of: 5′-GGA ACA GAC TGG GCT TTCATC-3′ (SEQ ID NO:12); 5′-GGA CAT CCC CTT GAC-3(SEQ ID NO:13); 5′-GTGGAT TCA CTT CTG CCA CG-3′ (SEQ ID NO:14); 5′-CTT CTG GCA TGC CCT ATGAG-3′ (SEQ ID NO:15); 5′-CAT GAC CCA GTT CGC CAT ATC CTG-3′ (SEQ IDNO:16); 5′-ATT CGT ATG CTG GAT CTC GCC ACC-3′ (SEQ ID NO:17). 5′-CGA ACGAAT CAT TCA GCA CCG-3′ (SEQ ID NO:44); 5′-CGG CAA TGT TTT ACT GTA GCGCC-3′ (SEQ ID NO:45); and complements thereof.
 8. A method foridentfying a beta-lactamase in a clinical sample, the method comprising:providing a pair of oligonucleotide primers specific for nucleic acidcharacteristic of the plasmid-mediated AmpC beta-lactamase enzymesdesigned as FOX-1, FOX-2, or MOX-1, wherein one primer of the pair iscomplementary to at least a portion of the beta-lactamase nucleic acidin the sense stand and the other primer of each pair is complementary toat least a portion of the beta-lactamase nucleic acid in the antisensestrand; annealing the primers to the beta-lactunase nucleic acid;simultaneously extending the annealed primers from a 3′ terminus of eachprimer to synthesize an extension product that is complementary to thenucleic acid strands annealed to each primer wberein each extensionproduct after separation from the beta-lactamase nucleic acid serves asa template for the synthesis of an extension product for the otherprimer of each pair; separating the amplified products; and analyzingthe separated amplified products for a region characteristic of thebeta-lactamase.
 9. The method of claim 8 wherein the primers areselected from the group of: 5′-CCA GCC GAT GCT CAA GGA G-3′ (SEQ IDNO:22); 5′-CAC GAA CGC CAC ATA GGC G-3′ (SEQ ID NO:23); and complementsthereof.
 10. A diagnostic kit for detecting a beta-lactmnase whichcomprises packaging, containing, separately packaged: (a) at least oneprimer pair capable of hybrid to beta-lactamase nucleic acid ofinterest, wherein the primers are specific for nucleic acidcharacteristic of the plasmid-mediated AmpC beta-lactamase enzymesdesignated as FOX-1, FOX-2, or MOX-1; (b) a positive and negativecontrol; and (c) a protocol for identification of the beta-lactamasenucleic acid of interest.
 11. The method of claim 10 wherein the primersare selected from the group of: 5′-CCA GCC GAT GCT CAA GGA G-3′ (SEQ IDNO:22); 5′-CAC GAA CGC CAC ATA GGC G-3′ (SEQ ID NO:23); and complementsthereof.
 12. A method for identifying an SHV family beta-lactamase in aclinical sample, the method comprsing: providing a pair ofoligonucleotide primers having the sequences 5′-TTA GCG TTG CCA GTG CTCG-3′ (SEQ ID NO:11), or a complement thereof, and 5′-GGG AAA CGG AAC TGAATG AG-3′ (SEQ ID NO:7), or a complement thereof, wherein one primer ofthe pair is complementary to at least a portion of the beta-lactamasenucleic acid in the sense strand and the other primer of each pair iscomplementary to at least a portion of the beta-lactamase nucleic acidin the antisense strand; annealing the primers to the beta-lactmaasenucleic acid; simultaneously extending the annealed primers from a 3′terminus of each primer to synthesize an extension product that iscomplementary to the nucleic acid strands annealed to each primerwherein each extension product after separation from the beta-lactamasenucleic acid serves as a template for the synthesis of an extensionproduct for the other primer of each pair; separating the amplifiedproducts; and analyzing the separated amplified products for a regioncharacteristic of the beta-lactamase.
 13. A diagnostic kit for detectinga beta-lactamase which comprises packaging, containing, separatelypackaged: (a) at least one primer pair capable of hybridizing tobeta-lactamase nucleic acid of interest, wherein the primers areselected from the group of 5′-TTA GCG TTG CCA GTG CTC G-3′ (SEQ IDNO:11), or a complement thereof, and 5′-GGG AAA CGG AAC TGA ATG AG-3′(SEQ ID NO:7), or a complement thereof, (b) a positive and negativecontrol; and (c) a protocol for identification of the beta-actamasenucleic acid of interest.